Abstract 054: Mechanical Stretch on Endothelial Cells Promotes Monocyte Differentiation Into Immunogenic Dendritic Cells

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Roxana Loperena ◽  
Wei Chen ◽  
Annet Kirabo ◽  
David G Harrison ◽  
Jose A Gomez
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Endothelial Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Fold Increase ◽  
Mechanical Stretch ◽  
T Cell Proliferation ◽  
Cyclical Stretch

We have shown that dendritic cells (DCs) from hypertensive mice and monocytes in humans accumulate highly reactive isolevuloglandins (isoLGs) or isoketals that adduct to protein lysines and promote T cell activation, specifically. Monocytes that traverse the endothelium have three different fates: reemerge into circulation as an activated monocyte; differentiate into macrophages or into monocyte-derived DCs. We hypothesized that human endothelial cells exposed to hypertensive mechanical stretch will promote conversion of human monocytes into immunogenic DCs. We co-cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the HAEC monolayer to either normal cyclical stretch (5%) or hypertensive uniaxial cyclical stretch (10%) for 48 hours. We found that co-culture of monocytes with HAECs exposed to 10% mechanical stretch markedly increased monocyte mRNA expression of the Th17 polarizing cytokines IL-6, I-23A and IL-1β and the p22 phox subunit of the NADPH oxidase compared to 5% stretch. HAECs exposed to 10% stretch promoted monocytes in culture to differentiate into DCs, as evidenced by the surface expression of DC-SIGN, CD83 and the co-stimulatory marker CD86. These monocytes also accumulated isoLG-modified proteins compared to controls (69.73 ± 5.802 vs 10.79 ± 1.854 respectively, p = 0.0001) as evidenced by intracellular staining and flow cytometry. We also co-cultured these monocytes with T cells from the same patient and examined proliferation of the latter using CFSE. Monocytes co-cultured with 10% stretched HAECs induced a 1,500-fold increase in CD4 + T cell proliferation and a 1,300-fold increase in CD8 + T cells proliferation. In addition, monocytes co-cultured with 10% stretched HAECs had a 2-fold increase in phosphorylated STAT3 expression compared to 5% stretch cultures. Conversely, monocytes-HAEC cultures exposed to 10% stretch and treated with STAT3 inhibitor, stattic, reduced their differentiation into DCs and prevented both CD4 + and CD8 + T cell proliferation. These data show that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote differentiation into activated DCs potentially via the STAT3 pathway.

Abstract 354: Mechanical Stretch on Endothelial Cells Promotes Monocyte Differentiation into Immunogenic Dendritic Cells

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Roxana Loperena ◽  
Wei Chen ◽  
Annet Kirabo ◽  
David G Harrison
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Fold Increase ◽  
Mechanical Stretch ◽  
T Cell Proliferation ◽  
Human Monocytes ◽  
Cyclical Stretch

Fatty acid oxidation leads to formation of highly reactive isoketals that adduct to protein lysines. We have shown that dendritic cells (DCs) from hypertensive mice accumulate isoketal-adducted proteins and promote T cell activation. In hypertension, the endothelium is activated to produce reactive oxygen species and to express adhesion molecules and chemokines that attract inflammatory cells. Human monocytes that traverse the endothelium differentiate into monocyte-derived DCs upon exposure to a pro-inflammatory state. We hypothesized that human endothelial cells exposed to mechanical stretch promote conversion of human monocytes into immunogenic DCs. We co-cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the HAEC monolayer to either normal cyclical stretch (5%) or hypertensive cyclical stretch (10%) using the Flexcell ® Tension System for 48 hours. We found that co-culture of monocytes with HAECs exposed to 10% mechanical stretch markedly increased monocyte mRNA expression of the Th17 polarizing cytokines IL-6, I-23A and IL-1β and monocyte chemokine CCL4 and the p22 phox subunit of the NADPH oxidase compared to 5% stretch. HAECs exposed to 10% stretch promoted monocyte in culture to differentiate into DCs, as evidenced by the surface expression of DC-SIGN, CD83 and the co-stimulatory marker CD86. These monocytes also had an accumulation of isoketal-adducted proteins compared to controls (69.73 ± 5.802 vs 10.79 ± 1.854 respectively, p = 0.0001) as evidenced by intracellular staining and flow cytometry. Monocytes co-cultured with 10%-stretched HAECs induced a 1,500-fold increase in CD4 + T cell proliferation and a 1,300-fold increase in CD8 + T cells proliferation as monitored by CFSE compared to 5% stretch controls. Conversely, monocytes-HAEC cultures exposed to 10% stretch and treated with STAT3 inhibitor, stattic, prevented both CD4 + and CD8 + T cell proliferation. These data show that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote their differentiation into immunogenic DCs potentially via the JAK/STAT3 pathway. These findings give insight into a new mechanism of lymphocyte activation in the vascular endothelium during hypertension.

scholarly journals Expression and functional significance of an additional ligand for CTLA-4

1993 ◽  
Vol 90 (23) ◽  
pp. 11054-11058 ◽  
Author(s):  
D J Lenschow ◽  
G H Su ◽  
L A Zuckerman ◽  
N Nabavi ◽  
C L Jellis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Costimulatory Molecule ◽  
T Cell Proliferation ◽  
Antigen Presenting

Effective T-cell activation requires antigen/major histocompatibility complex engagement by the T-cell receptor complex in concert with one or more costimulatory molecules. Recent studies have suggested that the B7 molecule, expressed on most antigen presenting cells, functions as a costimulatory molecule through its interaction with CD28 on T cells. Blocking the CD28/B7 interaction with CTLA4Ig inhibits T-cell activation in vitro and induces unresponsiveness. We demonstrate that another molecule(s), termed B7-2, is expressed constitutively on dendritic cells, is differentially regulated on B cells, and costimulates naive T cells responding to alloantigen. B7-2 is up-regulated by lipopolysaccharide in < 6 hr and is maximally expressed on the majority of B cells by 24 hr. In contrast, B7 is detected only on a subset of activated B cells late (48 hr) after stimulation. In addition, Con A directly induces B7-2 but not B7 expression on B cells. Finally, although both anti-B7 monoclonal antibodies and CTLA4Ig blocked T-cell proliferation to antigen-expressing B7 transfectants, only CTLA4Ig had any significant inhibitory effect on T-cell proliferation to antigens expressed on natural antigen presenting cells, such as dendritic cells. Thus, B7 is not the only costimulatory molecule capable of initiating T-cell responses since a second ligand, B7-2, can provide a necessary second signal for T-cell activation.

Abstract 069: Isoketals in Monocyte-Derived Dendritic Cells Activate T Cells and Promote Hypertension

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Roxana Loperena ◽  
Annet Kirabo ◽  
Sean S Davies ◽  
LJ Roberts ◽  
David G Harrison
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Oxidant Stress ◽  
Fold Increase ◽  
Human Monocytes ◽  
Pre Treatment

Fatty acid oxidation leads to formation of highly reactive γ-ketoaldehydes termed isoketals that adduct to protein lysines. We have shown that hypertension causes isoketals to accumulate in dendritic cells (DCs) and activate T cells. Human monocytes traversing the endothelium differentiate into DCs expressing CD83, MHC-II, and CD11c. We hypothesized that oxidative stress catalyzes the formation of isoketals which alters monocyte and DC function to promote monocyte transformation to DCs and T cell activation. Thus, we co-cultured human monocytes with aortic endothelial cells exposed to either a hypertensive 10% stretch or a normotensive 5% stretch for 48 hours using the Uniflex® culture system. We used flow cytometry to detect human DC markers (CD14-/CD83+) in monocytes exposed to stretch. We detected conversion of CD14-/CD83+ from CD14+ human monocytes and found they contained increased levels of isoketal-ligated proteins in the 10% compared to 5% stretch (77.47 ± 7.3 vs. 12.14 ± 3.5). We also found a 1.8 fold increase of the CD86 activation marker compared to controls. Exposure of murine monocyte-derived DCs to tert-butyl hydroperoxide (t-BHP) led to a 3-fold increase in isoketals and a 2-fold increase in CD86 in the CD11b+/CD11c+ population compared to controls. Isoketal formation in DCs exposed to t-BHP was scavenged with pre-treatment of 2-hydroxybenzylamine (2-HOBA) in vitro. DCs treated with t-BHP were co-cultured with T cells for 7 days. This promoted T cell survival and proliferation of CD8+ and CD4+ T cells compared to untreated controls; this effect was attenuated with pre-treatment of 2-HOBA. Moreover, adoptive transfer of t-BHP treated DCs to normal mice elevated the hypertensive response to a generally subpressor dose of angiotensin-II (128 ± 0.80 vs. 114 ± 0.48 in controls). We conclude that oxidant stress and isoketal formation in murine DCs promote T cell activation and hypertension. We hypothesize that exposure to hypertensive stretch in human endothelial cells transfers an oxidant signal to monocytes and promotes their transformation to DCs, which mature and promote an immune response. These findings provide a mechanism as to how T cells are activated in hypertension and provide insight into the inflammatory nature of this disease.

Functional IDO Is Expressed on CD34+- and Monocyte-Derived Dendritic Cells According to Differentiation Status.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1552-1552
Author(s):  
Antonio Curti ◽  
Sara Trabanelli ◽  
Chiara Onofri ◽  
Sergio Rutella ◽  
Raimondo De Cristofaro ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
T Cell Proliferation ◽  
Tnf Alpha ◽  
Gm Csf ◽  
Cytokine Cocktail ◽  
Cd34 Cells

Abstract Indoleamine 2,3-dioxigenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expression by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs). Although human monocyte-derived dendritic cells (Mo-DCs) have been shown to express IDO, little is known about the expression of IDO in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs) In the present study, we performed a full characterization of IDO expression and function by human dendritic cells including Mo-DCs and CD34+-derived DCs. Mo-DCs were generated from purified CD14+ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-gamma and a cytokine cocktail (IL-1beta, TNF-alpha, IL-6, PGE2). CD34+-derived DCs were generated from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-alpha, followed by 7 day-treatment with GM-CSF and IL-4 and then matured with the same stimuli used for MoDCs. After culture, DCs were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogenic proliferation and Tregs induction. Monocytes lack IDO expression. Immature Mo-DCs have little if any expression of IDO. During maturation, the cytokine cocktail is the most effective in up-regulating IDO, both at mRNA and protein level, which is paralleled with higher kynurenine production and inhibition of allogeneic T cell proliferation. PGE2 has a crucial role in inducing IDO expression, while IL6 has an opposite effect. Mature Mo-DCs are shown to generate a population of CD4+CD25+FOXP3+ Tregs which are capable of suppressing allogeneic T-cell proliferation. This inhibitory effect is abrogated by the addition of the IDO inhibitor 1-methyl tryptophan (1-MT). CD34+ cells lack IDO mRNA expression. During DC differentiation, IDO expression is observed at day 7, but not at day 14. Flow cytometry analysis of day 7 cells reveal a population of CD34−, CD14+, CD1a+/− cells. Similarly to Mo-DCs, maturation stimuli induce a marked up-regulation of IDO mRNA. Similar results are observed when IDO protein is measured. Production of kynurenine, inhibition of allogeneic T cell proliferation and generation of Tregs from normal CD3+ T-cells are also paralleled with IDO expression. In conclusion, in human DCs the maturation status is associated with a differential expression of IDO both in Mo-DCs and CD34+-derived DCs. Differentiation of CD34+ cells into DCs results in a transient and early expression of IDO at precursor level. Given its functional capacity to inhibit T-cell-mediated immune response, the expression of IDO along DC differentiation may indicate a protective role of the pool of precursor cells which are committed to DC lineage.

LFA-1 activity state on dendritic cells regulates contact duration with T cells and promotes T-cell priming

Blood ◽  
2010 ◽  
Vol 116 (11) ◽  
pp. 1885-1894 ◽  
Author(s):  
Sandra Balkow ◽  
Stefanie Heinz ◽  
Patricia Schmidbauer ◽  
Waldemar Kolanus ◽  
Bernhard Holzmann ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Contact Duration ◽  
Interacting Protein

Abstract A key event in the successful induction of adaptive immune responses is the antigen-specific activation of T cells by dendritic cells (DCs). Although LFA-1 (lymphocyte function–associated antigen 1) on T cells is considered to be important for antigen-specific T-cell activation, the role for LFA-1 on DCs remains elusive. Using 2 different approaches to activate LFA-1 on DCs, either by deletion of the αL-integrin cytoplasmic GFFKR sequence or by silencing cytohesin-1–interacting protein, we now provide evidence that DCs are able to make use of active LFA-1 and can thereby control the contact duration with naive T cells. Enhanced duration of DC/T-cell interaction correlates inversely with antigen-specific T-cell proliferation, generation of T-helper 1 cells, and immune responses leading to delayed-type hypersensitivity. We could revert normal interaction time and T-cell proliferation to wild-type levels by inhibition of active LFA-1 on DCs. Our data further suggest that cytohesin-1–interacting protein might be responsible for controlling LFA-1 deactivation on mature DCs. In summary, our findings indicate that LFA-1 on DCs needs to be in an inactive state to ensure optimal T-cell activation and suggest that regulation of LFA-1 activity allows DCs to actively control antigen-driven T-cell proliferation and effective immune responses.

scholarly journals ICOS-LICOS interaction is critically involved in TGN1412-mediated T-cell activation

Blood ◽  
2012 ◽  
Vol 119 (26) ◽  
pp. 6268-6277 ◽  
Author(s):  
Sabrina Weissmüller ◽  
Linda Y. Semmler ◽  
Ulrich Kalinke ◽  
Stefan Christians ◽  
Jan Müller-Berghaus ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Umbilical Vein ◽  
T Cell Proliferation ◽  
Cell Contact

TGN1412, a superagonistic CD28-specific antibody, was shown to require Fc-cross-linking or immobilization as a prerequisite to mediate T-cell proliferation and cytokine release in vitro. We used primary human umbilical vein endothelial cells (HUVECs) to study their ability to induce activation of TGN1412-treated T cells. We confirmed that peripheral primary human T cells do not show activation upon stimulation with soluble TGN1412 alone. Nevertheless, cocultivation of TGN1412-treated T cells with HUVECs induced T-cell activation that was further enhanced using cytokine prestimulated HUVECs. Unexpectedly, Fc-FcγR interaction was dispensable for endothelial cell–mediated proliferation of TGN1412-treated T cells. Transwell-culture assays showed that TGN1412-treated T cells need direct cell-to-cell contact to HUVECs to induce proliferation. We found that costimulatory ICOS-LICOS interaction between T cells and endothelial cells is critically involved in TGN1412-mediated effects. Blocking LICOS reduced TGN1412-mediated T-cell proliferation significantly, whereas recombinant LICOS fully conferred TGN1412-mediated T-cell proliferation. Of note, cytokine stimulation enhanced LICOS expression on HUVECs and ICOS-LICOS interaction up-regulated ICOS expression on TGN1412-treated T cells. Hence, we provide a model of positive feedback conferred by ICOS-LICOS interaction between TGN1412-treated T cells and endothelial cells.

scholarly journals Costimulation by B7 Modulates Specificity of Cytotoxic T Lymphocytes: A Missing Link That Explains Some Bystander T Cell Activation

1997 ◽  
Vol 186 (10) ◽  
pp. 1787-1791 ◽  
Author(s):  
Pan Zheng ◽  
Yang Liu
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Influenza Virus ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366–374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide–pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.

Abstract 13287: Dendritic Cells Play a Critical Role in the Initiation of Atherosclerosis

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Mengyao Jin ◽  
Peng Liu
Keyword(s):  
Cell Proliferation ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Critical Role ◽  
Cell Interaction ◽  
Plaque Formation ◽  
T Cell Proliferation ◽  
Control Group

Introduction: Dendritic cells (DCs) that are known as professional antigen-presenting cells have been found to pre-locate in non-inflammatory arterial wall and increasingly accumulate during atherosclerosis progression. Previous findings suggested that residential DCs in the intima are responsible for capturing modified lipids and forming foam cells during the initiation of atherosclerosis. Hypothesis: DC accumulation and enhanced DC-T cell interaction play a critical role in the initiation of atherosclerosis. Methods: We measured plaque formation, vascular DC accumulation and antigen-specific T cell proliferation mediated by isolated aortic cells in ApoE-/- mice, as well as DTR-CD11c/ApoE-/- or DTR-CD11b/ApoE-/- mice for conditional depletion of DCs or macrophages, respectively. A brief high-fat diet for 10 days was used as a model of initial atherosclerosis. Results: In addition to increased intimal DC accumulation and plaque formation in aortic roots, 10 days of HFD induced T cell infiltration in ApoE-/- mice, compared to those without HFD as the control. Isolated aortic cells from mice with 10-day HFD showed stronger capability in inducing antigen-specific T cell proliferation, compare to the control (HFD: 3.14±0.71%; no HFD: 1.56±0.36%; p=0.022). Single diphtheria toxin (DT) injection at day 1 yielded approximately 50% decrease in intimal DC accumulation, as well as 60% attenuation in plaque formation in DTR-CD11c/ApoE-/- mice after 10-day HFD. Capability of stimulating antigen-specific T cell proliferation was also impaired in aortic cells from DC-depleted mice (DT-treated: 1.62±0.30%; PBS-treated: 3.04±0.59%; p= 0.004), along with reduction in indirect conduction of T cell activation. In contrast, no significant changes were found in plaque formation and DC accumulation in DT-injected DTR-CD11b/ApoE-/- mice after 10 days of HFD, compared to control group. Furthermore, depletion of CD11b+ macrophages in either aortas or spleens didn’t alter capability of inducing antigen-specific T cell proliferation in DT-injected mice. Conclusions: These results suggested that vascular DCs rather than macrophages play a more important role in T cell activation and initiation of atherosclerosis.

scholarly journals Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro

2020 ◽  
Vol 11 ◽  
Author(s):  
Christian Binder ◽  
Felix Sellberg ◽  
Filip Cvetkovski ◽  
Erik Berglund ◽  
David Berglund
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mixed Lymphocyte Reaction ◽  
T Cell Proliferation ◽  
Allogeneic Mixed Lymphocyte Reaction ◽  
Dependent Cell

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

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