bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of mouse FoxP3+CD4+CD25+ regulatory T cells with an easy one-step flow cytometry analysis (89.17)
Authors: Qin Fenghua , Shin Jeffrey , Wei Bingyuan , Yang Xifeng , Kraft Brad , Lay Gene 
Published: January 2023

Abstract
Generally, CD4+CD25+FoxP3+ Tregs are characterized by cell surface staining with anti-CD25 and anti-CD4 antibodies, followed by intracellular staining with anti-FoxP3, which requires two steps. To simplify the characterization procedure, we developed a kit for labeling both surface and intracellular antigens of Tregs in one step. Mouse splenocytes were fixed and permeabilized at various fixing conditions: 1% paraformaldehyde (PFA), 0.5% PFA, 20% Ethanol, 70% Ethanol, BioLegend FoxP3 Permeabilization (Perm)/ Fixation buffer, or FoxP3 Perm/Fixation buffer with 0.01% Saponin. Then anti-mouse CD25 (mCD25), anti-mouse CD4 (mCD4), and anti-mouse FoxP3 (mFoxP3) antibodies were added to the splenocytes in one step. The cells were analyzed by flow cytometry after incubation for 20 minutes. Different fluorochrome combinations with anti-mCD25, anti-mCD4, and anti-mFoxP3 were tested. The results showed that anti-mFoxP3 Alexa Fluor 488 with the combinations of anti-mCD25-PE and anti-mCD4 APC produce comparable results to two-step staining in terms of percentage of CD4+CD25+FoxP3+ cells, absolute cell number, and fluorescence intensity. Among all these fixation conditions, the BioLegend FoxP3 Perm/ Fixation buffer yields the best results. Our development of a one step kit provides a fast, dependable new tool for evaluating and exploring effects of CD4+CD25+FoxP3+ Tregs in mice.
SHP-1 influences resistance of CD4+ T cells to regulatory T cell suppression (89.25)
Authors: Sankarshanan Mohan , Iype Tessy , Mauldin Ileana , Lorenz Ulrike M 
Published: January 2023

Abstract
The mechanistic and molecular understanding about factors that regulate the susceptibility and/or resistance of T cells to suppression by regulatory T cells (Treg) is very limited. The protein tyrosine phosphatase SHP-1 is a negative regulator of TCR-mediated signaling. SHP-1-deficient T cells hyper-proliferate in response to antigen stimulation. We therefore asked whether SHP-1 affects the susceptibility of CD4+ T cells to suppression by Treg cells. Our in vitro suppression assays showed that heterozygous (me/+) and homozygous (me/me) SHP-1-deficient CD4+ T cells were more resistant to Treg cell-mediated suppression than +/+ CD+ T cells, indicating that SHP-1 modulates the resistance to Treg cell suppression.
In order to gain a better understanding of the mechanism that controls the resistance to suppression, we examined the conjugate formation between CD4+ T cells and the antigen presenting cells (APCs). Using flow cytometric and ImageStream100 (Amnis) analyses, we observed that me/me and me/+ T cells form more conjugates with APCs than +/+ T cells both in the presence or absence of Treg cells. Moreover, +/+ T cells treated with an inhibitor of SHP-1 showed a comparable increased conjugate formation as SHP-1-deficient T cells indicating that the observed phenotype is T cell intrinsic and not due to developmental differences. Furthermore, SHP-1-deficient T cells exhibited elevated surface levels of ICAM-1. Ongoing studies are addressing whether ICAM-1 directly affects the resistance to suppression. Taken together, the increased resistance to suppression displayed by SHP-1-deficient T cells and the enhanced capability of forming conjugates with APCs compared to wild type T cells suggest that the ability of forming T cell-APC conjugates influences to the susceptibility and/or resistance of a T cells to suppression.
Source: NIH, RO1 AI 048672
Regulatory T cell enrichment by Adenosine A3 receptor agonist is mediated through PLD signal Inhibition (89.28)
Authors: CHANDRASEKARAN UMA , IWASHIMA MAKIO 
Published: January 2023

Abstract
Phospholipase D (PLD) is a phosphodiesterase that catalyzes the conversion of phosphatidyl choline (PC) to phosphatidic acid (PA). We have previously demonstrated that inhibition of PLD signaling leads to preferential expansion of naturally occurring Tregs. Here, we show that adenosine blocks PLD signaling in activated T cells and causes increased frequency of nTregs in vitro. Adenosine (Ado), a naturally arising nucleoside has been implicated to be immunosuppressive but the mechanisms involved in immune suppression are not clear.Ado is released from dead cells due to nucleotide degradation. It is also produced by hydrolysis of ATP by CD39/CD73. Ado elicits intracellular responses by activating G-protein coupled receptors (A1, A2a, A2b, and A3). Increased concentrations of Ado have been detected in the individuals with the defect in ecto-adenosine deaminase activity (ADA and CD26), organs with high level of cell death (e.g. thymus, placenta), or tissues under hypoxic conditions (e.g. large solid tumors). Increase of Ado concentration was associated with severely compromised immune responses.
T cells express two types of adenosine receptors, namely A2a and A3. Our data show that agonist specific to Adenosine A3 receptor blocks expansion of non-regulatory T cells by suppressing TCR mediated signaling events and reduction of cytokine production. However, expansion of CD4+CD25+ naturally arising regulatory T cells (nTregs) was not affected by A3 agonist. Thus, A3 mediated signaling may contribute to establish reduced immune responses in the Ado rich environment by changing the balance between effector and regulatory T cells.
Characteristics of CD8+CD25- cells induced to become suppressor cells ex-vivo with IL-2 and TGF-β (89.34)
Authors: Lu Ling , Wang Julie , Wang Xuehao , Zhang Feng , Lo Jiwen , Brand David , Horwitz David , Zheng Song Guo 
Published: January 2023

Abstract
CD8+ cells stimulated with IL-2 and TGF-β can be induced to develop suppressive activity. However, the phenotype and functional characteristics of these polyclonal regulatory T cells (iTregs) have not been well characterized. To distinguish expanded thymus-derived Foxp3+ CD8+ cells from conventional CD8+CD25- cells induced to become Foxp3+ Tregs, we used Foxp3 GFP transgenic mice. Splenic GFP- cells from these mice were stimulated with anti-CD3/28 coated beads. Following 4-day stimulation, 35% of CD8+ cells become CD25+GFP+ cells in the presence of IL-2 and TGF-β. Granzyme B expression was decreased compared to CD8+ cells activated without TGF-β. These cells were also anergic following restimulation. Although their Foxp3 expression is lower than TGF-β -induced CD4+ cells, they displayed similar suppressive activity in cell contact-dependent, non-cytotoxic suppressive activity manner in a standard in vitro assay. Neutralization of TGF-β, IL-10 or IL-10 receptor and the TGF-β receptor I (ALK5) inhibitor was unable to abolish the suppressive activity. Similar to CD4+ iTregs, adoptive transfer of CD8+ iTregs suppressed a chronic GVHD with a lupus-like syndrome. Thus, TCR stimulation with IL-2 and TGF-β can induce CD8+ cells as well as CD4+ cells to become Foxp3+ Treg cells and both of these subsets have protective effects against pathologic immune-mediated inflammation. (Supported by ACR REF Award and Outstanding youth Scientist Award from NSFC)
Characterization of Thymic Regulatory T cell development (89.6)
Authors: Hsieh Chyi S 
Published: January 2023

Abstract
The development of Foxp3+ CD4+ regulatory T (Treg) cells in the thymus is important for the preservation of self-tolerance and the prevention of spontaneous autoimmunity. It has been suggested that the CD4+CD8+ double positive (DP) stage is the earliest that Foxp3+ expression can be observed. We hypothesized that the antigen specificity of Foxp3+ DP cells may differ from Foxp3+ CD4+CD8- (CD4SP) cells due to the different antigenic repertoires presented by cortical thymic epithelial cells versus medullary APCs. However, purification of Foxp3+ DP cells proved difficult as many of those events on flow cytometry represented doublets of Foxp3+ CD4SP cells and Foxp3- DP cells. Our data therefore suggest that the frequency of Foxp3+ cells that can be found at the DP stage is around 1%, which is much smaller than previously reported. Rather, our current data suggests that thymic Treg cell development occurs primarily at the HSAhi CD4SP stage involving a two-step process involving both TCR-instructive and TCR-independent steps.
Tim-3 Regulation of T Cell Effector Function (90.13)
Authors: Su Ee W , Lee Judong , Kane Lawrence P 
Published: January 2023

Abstract
Tim-3 is a type I transmembrane glycoprotein first identified through a screen for TH1-specific cell surface markers. Since then, it has been detected on a wide variety of cell types. Galectin-9, a β-galactoside binding lectin is the only known ligand of Tim-3 and has been used to study the function of Tim-3 due to the lack of agonistic Tim-3 antibodies. Galectin-9 induces apoptosis and calcium flux in TH1 cells but promotes the maturation of immature monocyte-derived dendritic cells and induces IgE-sensitized mast cells to secrete higher levels of TH2 cytokines. To better understand the regulation of T cell effector function by Tim-3, we treated TH1 cells with varying doses of galectin-9 and found that at an optimal concentration, galectin-9 has the ability to enhance IFN-γ production by TH1 cells without affecting their viability. Then, using luciferase reporter assays, we found that overexpression of Tim-3 in Jurkat T cells can enhance NF-κB and NFAT/AP-1 activity downstream of the T-cell receptor and CD28. Therefore, these findings indicate that Tim-3 can positively affect T cell effector function.
Effect of ROR γt on regulation of IFN-γ secretion of T cell subpopulations (90.15)
Authors: LI Baiqing , XUE Zhuping , Ma Fei 
Published: January 2023

Abstract
It has been known that retinoid-related orphan receptor γt (RORγt) is a key transcription factor for development of IL-17 producing CD4 T cells (Th17), which are negatively regulated by interferon-γ (IFN-γ) produced by Th1 cells, however, whether the RORγt regulates IFN-γ production in T cell subsets has not been elucidated. In our study the peripheral blood mononuclear cells isolated from peripheral blood of healthy human donors were stimulated with CD3 mAB and cultured in IL-2 containing medium for 12 days. The activated T cells were transfected with three chemical synthesized small interfering RNA (siRNA) sequences for RORγt gene using liposomes. By using semi-quantity RT-PCR assay, the RORγt gene expression was inhibited in activated T cells that transfected with RORγt-siRNA-1026 and -1197, but was not inhibited in that with RORγt-siRNA-982. By using flowcytometry, the percentages of IFN-γ producing cells in total T cells, CD4+ T cells and CD8+ T cells that transfected with the sequence of RORγt-siRNA-1026, significantly decreased to 35.19, 32.78, and 41.84, respectively, in comparison of nontransfected group (43.39, 44.54, and 50.04, respectively) (p&lt; 0.05). In conclusions, the transcription factor RORγt may also be involved in the positive regulation for IFN-γ production in human T cells, especially in CD4+ T cells.
Cytotoxicity of human CD8+ T-cells and NK-cells is suppressed by an alternative NKG2D splice variant (90.19)
Authors: Karimi Mobin  A , Bachmann Michael H , Cao Thai M , wang Chao , Kim Edward , Watts Tania H , Negrin Robert S , Zhang Li 
Published: January 2023

Abstract
NKG2D is an activating receptor crucial to diverse aspects of immune function. NKG2D-mediated cytotoxicity is utilized by human natural killer (NK) cells and CD8+ T-cells. Here, we functionally characterize an alternative splice variant of NKG2D in non-activated and activated human CD8+ T-cells, which is expressed as a membrane-anchored truncated protein that lacks the ligand-binding ectodomain. We found this truncated NKG2D (T/NKG2D) isoform to be unable to transmit an activating signal for cytolysis and its enforced expression to inhibit killing mediated by full-length NKG2D (F/NKG2D). Abundance of T/NKG2D transcripts is variable in non-activated and activated CD8+ T-cells, and inversely correlates with killing capacity. Co-immunoprecipitation showed that T/NKG2D pairs with DAP10 and heterodimerizes with F/NKG2D, suggesting that dominant negative regulation is due to formation of heterodimeric NKG2D receptor complexes incapable of binding to its ligands. Thus, competitive inhibition through the alternatively spliced NKG2D variant may constitute a novel mechanism that regulates NKG2D-mediated signaling and cytotoxicity in NK and CD8+ T-cells.
Distinct subsets of human skin dendritic cells differ on their ability to initiate Th17 responses (90.24)
Authors: Mathers Alicia R , Janelsins Brian M , Rubin Joseph P , Tkacheva Olga A , Shufesky William J , Morelli Adrian E , Larregina Adriana T 
Published: January 2023

Abstract
Activated human cutaneous dendritic cells (DCs) have the ability to initiate Th1 and Th2 immunity. However, whether human cutaneous DCs are capable of biasing pro-inflammatory Th17 responses remains little understood. Utilizing human skin explants composed by both epidermis and dermis or epidermal or dermal sheets to collect activated skin-migratory DCs (smiDCs), we demonstrated that smiDCs stimulate allogeneic naïve CD4+ T cells to differentiate simultaneously into two distinct effector Th17 and Th1 populations with ability of skin homing and inducing severe tissue damage. Of the two main myeloid DC populations resident in the skin, the subset of epidermal skin-migratory Langerhans cells (smiLCs) were capable of inducing Th17 responses. This effect depended on the combined effects of IL-15 and stabilized IL-6 trans-signaling of naïve CD4+ T cells. Purified skin-migratory DDCs did not synthesize IL-15 and were unable to bias Th17 responses however, they acquired the ability to bias Th17 cells in co-cultures with CD4+ T cells supplemented with IL-15 and stabilized IL-6. Overall, our data demonstrate that human cutaneous LCs induce Th17 responses by mechanisms different from those previously described for mouse DCs and human monocytes and highlight the need to target clinical treatments based on these variations.
Supported by NIH grants: R01 CA100893 (ATL)
In vivo opposing regulation of vasoactive intestinal peptide receptor-1 and -2 throughout the T cell immune response (90.25)
Authors: Vomhof-DeKrey Emilie E , Haring Jodie , Dorsam Glenn P 
Published: January 2023

Abstract
This study investigates vasoactive intestinal peptide receptor 1 (VPAC1) and VPAC2 regulation during the immune response of murine T cells. VPAC1 is an anti-proliferative, G-protein coupled receptor that has been shown to be downregulated upon ex vivo T cell activation, whereas VPAC2 was shown to be upregulated. To date, regulation of VPAC1 and 2 have not been elucidated in primary, murine T cells throughout the phases of a T cell immune response following infection and whether they have a role in memory T cell evolution. Splenic CD4 T cells were isolated from C57Bl/6J mice 24hr post in vivo activation with anti-CD3. Flow cytometry (Accuri) analysis confirmed 90% activation of cells (CD69+/CD25+). QPCR analysis of total RNA showed VPAC1 levels decreased 93% in activated CD4 T cells, while VPAC2 levels increased 66%, possibly suggesting that these receptors regulate early components of the CD4 T cell response. These results prompted us to pursue an in vivo activation model utilizing OT-I and OT-II T cells. We hypothesize in CD4 T cells, VPAC1 levels will decrease upon activation and gradually increase to naïve levels throughout contraction into memory phase, while VPAC2 levels will increase upon activation and gradually decrease to lower naïve levels. The reciprocal expression pattern of these receptors may indicate they have opposing functions in regulating the kinetics of the T cell response and memory cell evolution.
1KO1 DK064828, COBRE- 2P20RR015566
CD70 drives the proliferation of memory CD8 T cells during secondary responses (90.5)
Authors: Al-Shamkhani Aymen , Taraban Vadim Y , Buchan Sarah L 
Published: January 2023

Abstract
Recent studies have either showed CD27 to be necessary or unnecessary for memory CD8 T cell responses. Heterogeneity in memory CD8 T cells in their ability to respond to CD27 signaling and/or differential use of CD27 ligand (CD70) by different dendritic cell subsets may explain the variable dependence on CD27 in different models. To distinguish between these two possibilities, we used soluble recombinant CD70 in vivo in order to overcome the requirement for endogenous CD70 expression. We show that secondary expansion and function of central and effector memory CD8 T cells are boosted by administration of soluble CD70. In vivo BrdU incorporation studies demonstrated increased proliferation of CD8 memory T cells in mice that had received soluble CD70, compared with those that received antigen alone. Furthermore, activation of CD27-low effector memory CD8 T cells led to CD27 upregulation to a level similar to that expressed by central memory T cells. We conclude that optimal proliferation of memory CD8 T cells requires a costimulatory signal via CD27 and that both central and effector memory CD8 T cells are equally competent in responding to CD27 ligation by CD70. Therefore, discrepancies in the requirement for CD70 during secondary responses reported in previous studies are likely to be due to differential use of CD70 by alternative antigen presenting cells. This work is funded by Cancer Research UK.
A novel monoclonal antibody that identified a transmembrane isoform of CD160 (CD160-TM) selectively expressed on human NK cell upon activation (90.9)
Authors: Bensussan Armand , Giustiniani Jerome , Marie-Cardine Anne 
Published: January 2023

Abstract
CD160 has been initially identified as a GPI-anchored MHC-class I activating receptor mainly expressed on peripheral blood NK cells. Here, we report the identification of an additional CD160-related mRNA generated through alternative splicing of CD160 gene, which encoded a putative CD160 transmembrane isoform (CD160-TM). We first establish that CD160-TM surface expression is highly restricted to NK lymphocytes and is activation-dependent. In addition, we provide evidences that CD160-TM represent a novel activating receptor, as assessed by the increased CD107a NK cell surface mobilization observed upon its engagement with a specific monoclonal antibody. Finally, we study the activating signaling obtained following CD160-TM engagement. The identification of CD160-TM therefore provides new possibilities regarding the role of CD160 isoforms in the regulation of NK cell functions.
DAP12 renders liver dendritic cells resistant to maturation (91.14)
Authors: Sumpter Tina L , Thomson Angus W 
Published: January 2023

Abstract
Liver APC are constitutively exposed to gut-derived LPS, yet are refractory to LPS-induced maturation. We and others have hypothesized that resistance to LPS in liver DC reflects molecular inhibition of NF-κB activation. DNAX-activating protein of 12kDa (DAP12), a transmembrane adaptor protein, dampens co-stimulatory molecule and cytokine expression associated with inhibition of NF-κB in myeloid (m)DC. In this study, we evaluated the function of DAP12 in mDC (CD11c+CD11b+NK1.1-B220-) purified from livers or spleens of Fms-like tyrosine kinase 3 ligand (10 μg/day/10d)-mobilized C57BL/10 mice (in which DC populations were enriched), then cultured overnight with LPS. To test the hypothesis that DAP12 impairs LPS-induced maturation of liver mDC, DAP12 was silenced with siRNA (400nM) 2h prior to LPS stimulation. Liver mDC did not upregulate CD80, CD86, B7-H1 (PD-L1), B7RP or IL-12 to the extent of splenic mDC in response to LPS. Silencing DAP12 enhanced expression of CD80, CD86, IL-12 and the Th1-polarizing molecule, Delta4, but not B7-H1 or B7RP in liver and spleen mDC in response to LPS. These effects were more pronounced in liver mDC. We conclude that the comparatively immature state of liver mDC may be regulated, in part, by DAP12. Supported by American Liver Foundation and American Transplantation Society Fellowships and NIH RO1AI60994.
Estrogen receptors promote langerin+ dendritic cell differentiation and migration from skin during inflammation in vivo (91.3)
Authors: Bajana Sandra , Agrawal Hemant , Carreras Esther , Kovats Susan 
Published: January 2023

Abstract
CD207/langerin+ dendritic cells in the epidermis (eLC) and dermis (dLC) initiate immune responses in the skin. We previously published that estrogen receptor alpha (ERα) signaling promotes the differentiation of langerin+ DC in a GM-CSF-driven culture model. To compare the efficiency of dLC differentiation from ERα+ and ERα-/- bone marrow (BM) cells in vivo, we generated mixed competitive ERα+/ERα-/- BM chimeric mice. In the radiation-induced inflammatory environment early after reconstitution, dLC in cutaneous lymph nodes (cLN) were derived preferentially from ERα+ BM cells, indicating that during systemic inflammation ERα signaling promotes dLC differentiation from short term repopulating progenitors. To determine the relative ability of ERα+ and ERα-/- LC to become activated during local skin inflammation, green fluorescent dye in acetone/dibutylphthalate was applied to the skin of ERα+ and ERα-/- mice. Compared to ERα-/- mice, after 24 hr, ERα+ mice contained significantly greater numbers of langerin+ CD11b+ eLC that had acquired the green dye and migrated to cLN. Moreover, ERα+ mice contained higher numbers of new inflammatory CD11bhi CD11clo F4/80+ myeloid cells in the spleen. In sum, ERα signaling in response to endogenous estrogens promotes the development, activation and migration of skin-derived langerin+ DC during inflammation in vivo. Supported by OCAST HR06-157 & NIH AI079616, RR020143RR020143.
SHP-2 constitutively suppresses natural killer cell polarization and granule release in response to tumor target cells (91.4)
Authors: Purdy Amanda K , Campbell Kerry S 
Published: January 2023

Abstract
The tyrosine phosphatase, SHP-2, promotes the full activation of Ras/ERK in many cytokine and growth factor receptor pathways. In contrast, SHP-2 inhibits activation of natural killer (NK) cells by binding to inhibitory receptors, known as killer cell Ig-like receptors (KIRs). To determine how SHP-2 impacts NK cell activation pathways, we employed retroviral shRNA knockdown and over-expression strategies in human NK cell lines. In response to stimulation with tumor cells, SHP-2 shRNA expressing NK cells had elevated cytolytic activity and IFN-γ production, whereas cells over-expressing wild type or gain-of-function mutants of SHP-2 exhibited dampened activities. SHP-2 suppressed microtubule organizing center (MTOC) polarization and granzyme B release in a dose-dependent manner, but NK-tumor cell conjugation was not significantly affected. The suppressed NK cell function was independent of inhibitory KIR. In summary, our data demonstrate that SHP-2 constitutively suppresses NK cell activation in response to tumor cells. This suppression occurs at a stage after NK-tumor cell adhesion, but upstream of MTOC-based cytoskeletal rearrangement and granule release. These studies have identified a new, KIR-independent suppressive role for SHP-2 in regulating NK cell function and provide mechanistic insight for this role.
This work was supported by NIH grants CA083859 (K.S.C) and CA009035-32 (A.K.P).