scholarly journals Small-Molecule Antagonist of VLA-4 (GW559090) Attenuated Neuro-Inflammation by Targeting Th17 Cell Trafficking across the Blood-Retinal Barrier in Experimental Autoimmune Uveitis

2020 ◽  
Author(s):  
Yi-Hsing Chen ◽  
Malihe Eskandarpour ◽  
Xiaozhe Zhang ◽  
Grazyna Galatowicz ◽  
Greenwood John ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Th17 Cell ◽  
Th17 Cells ◽  
T Cell Subsets ◽  
Experimental Autoimmune Uveitis ◽  
Blood Retinal Barrier ◽  
Integrin Inhibitor ◽  
Experimental Autoimmune

Abstract Background: The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets.Methods: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry.Results: There was a significant reduction in clinical and histological scores in GW10 and Dex treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10 treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 by adhering to endothelial monolayers.Conclusions: This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

scholarly journals Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yi Hsing Chen ◽  
Malihe Eskandarpour ◽  
Xiaozhe Zhang ◽  
Grazyna Galatowicz ◽  
John Greenwood ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Th17 Cell ◽  
Th17 Cells ◽  
T Cell Subsets ◽  
Experimental Autoimmune Uveitis ◽  
Blood Retinal Barrier ◽  
Integrin Inhibitor ◽  
Experimental Autoimmune

Abstract Background The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. Methods Mice (female; B10.RIII or C57Bl/6; aged 6–8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. Results There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. Conclusions This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

Evidence for Associations Between Th1/Th17 “Hybrid” Phenotype and Altered Lipometabolism in Very Severe Graves Orbitopathy

2020 ◽  
Vol 105 (6) ◽  
pp. 1851-1867 ◽  
Author(s):  
Sijie Fang ◽  
Shuo Zhang ◽  
Yazhuo Huang ◽  
Yu Wu ◽  
Yi Lu ◽  
...  
Keyword(s):  
T Cell ◽  
Th17 Cell ◽  
Th17 Cells ◽  
T Cell Subsets ◽  
Th1 Cell ◽  
Effector T Cell ◽  
Cell Lineages ◽  
Graves Orbitopathy ◽  
Ifn Γ

Abstract Purpose The purpose of this article is to investigate the characteristics of Th1-cell and Th17-cell lineages for very severe Graves orbitopathy (GO) development. Methods Flow cytometry was performed with blood samples from GO and Graves disease (GD) patients and healthy controls, to explore effector T-cell phenotypes. Lipidomics was conducted with serum from very severe GO patients before and after glucocorticoid (GC) therapy. Immunohistochemistry and Western blotting were used to examine orbital-infiltrating Th17 cells or in vitro models of Th17 polarization. Results In GD, Th1 cells predominated in peripheral effector T-cell subsets, whereas in GO, Th17-cell lineage predominated. In moderate-to-severe GO, Th17.1 cells expressed retinoic acid receptor-related orphan receptor-γt (RORγt) independently and produced interleukin-17A (IL-17A), whereas in very severe GO, Th17.1 cells co-expressed RORγt and Tbet and produced interferon-γ (IFN-γ). Increased IFN-γ–producing Th17.1 cells positively correlated with GO activity and were associated with the development of very severe GO. Additionally, GC therapy inhibited both Th1-cell and Th17-cell lineages and modulated a lipid panel consisting of 79 serum metabolites. However, in GC-resistant, very severe GO, IFN-γ–producing Th17.1 cells remained at a high level, correlating with increased serum triglycerides. Further, retro-orbital tissues from GC-resistant, very severe GO were shown to be infiltrated by CXCR3+ Th17 cells expressing Tbet and STAT4 and rich in triglycerides that promoted Th1 phenotype in Th17 cells in vitro. Conclusions Our findings address the importance of Th17.1 cells in GO pathogenesis, possibly promoting our understanding of the association between Th17-cell plasticity and disease severity of GO.

scholarly journals JIA patient T cells differentiate into Th1, Th17 and Th1.17 effector cells under Th1 polarizing conditions

2021 ◽  
Author(s):  
Anna E Patrick ◽  
Tashawna Esmond ◽  
Kayla Shoaff ◽  
David M Patrick ◽  
David K Flaherty ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Cell Cultures ◽  
Th17 Cells ◽  
Mononuclear Cells ◽  
Healthy Children ◽  
Th1 Cell ◽  
Peripheral Blood Mononuclear ◽  
Ifn Gamma

Objective. T helper cells develop into discrete Th1, Th2 or Th17 lineages that selectively express IFN-gamma, IL-4/IL-5/IL-13, or IL-17, respectively and actively silence signature cytokines expressed by opposing lineages. Our objective was to compare Th1, Th2 and Th17 polarization in cell culture models using JIA patient samples. Methods. Peripheral blood mononuclear cells were isolated from JIA or healthy prepubescent children. T cell naive and memory phenotypes were assessed by flow cytometry. T cell proliferation was measured using a fluorescence-based assay. Th cell cultures were generated in vitro and IFN-gamma, IL-17, and TNF-alpha measured by ELISA and flow cytometry. Results. JIA Th1 cells produced increased IFN-gamma and inappropriately produced IL-17. JIA Th17 cells produced increased IL-17. JIA Th1 cell cultures develop dual producers of IFN-gamma and IL-17, which are Th1.17 cells. JIA Th1 cultures expressed elevated levels of both T-bet and ROR-gamma-T. RNA sequencing confirmed activation of immune responses and inappropriate activation of IL-17 signaling pathways in Th1 cultures. A subset of JIA patient samples was disproportionally responsible for the enhanced IFN-gamma and IL-17 phenotype and Th1.17 phenotype. Conclusions. This study reveals that JIA patient uncommitted T cell precursors, but not healthy children, inappropriately develop into inflammatory effector Th1.17 and Th17 cells under Th1 polarizing conditions.

Massive, Clinical Grade Expansion Of Polyclonal T Cells Using Blinatumomab For Adoptive Autologous Cellular Therapy Of CLL Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3272-3272 ◽  
Author(s):  
Josée Golay ◽  
Anna D’amico ◽  
Gianmaria Borleri ◽  
Maria Chiara Finazzi ◽  
Giulia Quaresmini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Peripheral Blood ◽  
T Cell Subsets ◽  
Th2 Cells ◽  
Adoptive Therapy ◽  
Specific Patient

Abstract Background The combined use of chemotherapy and monoclonal antibodies has proved highly effective for the treatment of CLL but often results in severe life threatening immunosuppression. The development of adoptive therapy with autologous T cells could be clinically relevant to overcome these problems. Methods We have devised a novel, simple and efficient method for ex vivo expansion of normal autologous T cells from the peripheral blood of CLL patients for adoptive therapy, using blinatumomab (CD3xCD19) and rhIL-2 in serum-free medium. The complete phenotype of in vitro expanded T cells was analyzed by flow cytometry and their cytotoxic activity by calcein release assays. Results We performed 18 expansions of T cells, starting from a very small volume of peripheral blood from untreated CLL patients (mean 10.3 ml, range 2-30 ml) that contained a mean of 9.2x106 T cells (range 0.4 to 51x106)(Fig.1). This method allowed reproducible expansion in about 21 days of a mean 410x106 CD3+ T cells (range 71 to 2184x106). The mean fold expansion of T cells in about 3 weeks of in vitro culture was 224 (range 4.4-1326). The only significant contaminant in final Blinatumomab Expanded T cell cultures (BET) were NK cells (mean 18.5%). Indeed addition of blinatumomab and rhIL-2 to the cultures led to a rapid decrease in CLL B cells, which took place from days 7 to 14 onwards and resulted in their complete depletion within 3 weeks (mean 0.2% CLL B cells at days 18-25). Only in one case, a significant percentage of CLL B cells could be observed at the end of culture, but this was due to the particularly high percentage neoplastic cells in the starting population in this patient (98%), resulting in relatively late depletion of these cells, which took place between days 14 and 21, and therefore remained detectable in this case at day 24 (3.8% CLL B cells at day 24). Despite the very low percentage of starting T cells in this specific patient (1.2%), 152x106 T cells could be obtained, equivalent to a 42 fold expansion. In the 18 expansions performed, the resulting BET cells contained both CD4+ and CD8+ cells in varying proportions (median 46.2% and 44.4% respectively). Only in two cases the final product was composed predominantly of CD4+ cells (95%). Expanded T cells were polyclonal, as shown by TCR Vβ expression which was within the normal range by flow cytometry. Indeed CMV specific clones, detected by CMV peptide (pp65495-503)-loaded HLA-A*0201 tetramer, were expanded using this method and detected in equivalent proportion before and after expansion. Final T cells were composed predominantly of the effector and central memory subsets. Th1 were slightly prevalent over Th2 cells (means 20% and 10%, respectively), whereas Th17 and Treg were less than 1%. Since CLL derived T cells have been shown previously to have enhanced expression of the synapse regulators CD272 and CD279 compared to normal T cells, leading to impaired immunological synapse formation, we have analyzed these markers in both starting and BET cells from 4 patients. We observed that CD272 and CD279 diminished in BET compared to the starting CLL T populations (from 73% to 19% and 61% to 18%, respectively). These data suggest that stimulation and expansion with blinatumomab and rhIL-2 has normalized expression of these regulators on CLL T cells. Indeed BET were highly cytotoxic against CD19+ targets cell lines or primary CLL cells, with 70-90% lysis at a 3:1 effector target ratio in presence of blinatumomab. Finally BET were compared to Xcellerated cells expanded using anti-CD3/CD28 Dynabeads and rhIL-2. The expansion protocols using either blinatumomab or anti-CD3/CD28 Dynabeads showed equivalent efficiency and comparable cell composition at the end of culture. Further comparison of the T cell subsets present in BET or CD3/CD28 cultures is in progress. Conclusions These data altogether suggest that the use of blinatumomab and rhIL-2 provides a reproducible, simple and GMP-compliant protocol, allowing expansion of large numbers of autologous polyclonal T cells depleted of CLL cells, from relatively small volumes of peripheral blood from CLL patients. This approach is an attractive option for adoptive therapy in these patients after immunosuppressive treatments. Disclosures: No relevant conflicts of interest to declare.

Pregnancy-induced fluctuations in functional T-cell subsets in multiple sclerosis patients

2010 ◽  
Vol 16 (9) ◽  
pp. 1073-1078 ◽  
Author(s):  
Rinze F Neuteboom ◽  
Evert Verbraak ◽  
Annet F Wierenga-Wolf ◽  
Marjan van Meurs ◽  
Eric AP Steegers ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
T Cell ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Post Partum ◽  
T Cell Subsets ◽  
Third Trimester ◽  
Treg Frequency ◽  
Post Partum Period

Background: During pregnancy, especially during the third trimester, multiple sclerosis (MS) disease activity is reduced. It is not known which factors mediate this disease amelioration. Objective: To study whether the frequency of two important T-cell subsets, T-helper 17 (Th17) and regulatory T-cells (Treg), is altered in relation to pregnancy-induced MS disease amelioration. Methods: Each individual was tested longitudinally, after sampling of blood at timepoints before pregnancy, during the first and third trimester, and in the early post-partum period. Frequencies of Th17 cells were assessed after short (4 hours) re-stimulation of peripheral blood lymphocytes with PMA and ionomycin, followed by flow cytometry using CD4, CD45RO and IL-17A antibodies. To assess peripheral blood Treg frequencies, we used six-colour flow cytometry with antibodies against CD3, CD4, CD25, CD127, FoxP3 and HLA-DR, to specifically identify Treg. Results: Both MS patients ( n = 9) and controls ( n = 8) displayed unaltered Th17 frequencies during pregnancy. In contrast, circulating Treg frequency significantly decreased in MS patients ( n = 15) during the first and third ( p < 0.001) trimesters compared with the period before pregnancy. In the post-partum period, the frequency of circulating Treg again resurged back to near pre-pregnancy levels. In controls ( n = 15) comparable frequency kinetics were observed in that post-partum a significant increase in circulating Treg frequency was detected compared with the first ( p < 0.001) and third ( p = 0.012) trimester. Conclusions: Third trimester amelioration is not related to the fluctuation of circulating Th17 cells. Furthermore, a paradoxical decrease of immunosuppressive circulating Tregs can be observed during this phase, both in MS patients and controls.

The Antioxidant n-Acetylcysteine Blocks Direct Regulatory T Cell Mediated Suppression of CD4+ Effector T Cells In Vitro

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2765-2765
Author(s):  
Todd W. Kelley ◽  
Olga Efimova
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Effector T Cells ◽  
T Cell Subsets ◽  
Potential Contribution ◽  
Tgf Beta ◽  
Treg Suppression ◽  
Dose Dependent

Abstract Abstract 2765 Background: CD4+CD25+FOXP3+ regulatory T cells (Tregs) employ a range of suppressive strategies including factors that are cytotoxic to target CD4+CD25−FOXP3− effector T cells (Teff) such as perforin and granzyme B and those that suppress proliferation, differentiation and/or cytokine production including TGF-beta1, IL-10 and CTLA-4. The relative contribution of each of these mechanisms to Treg function is unclear but from the available data their importance appears context specific. Because T cells are very sensitive to the redox state of the microenvironment, and display a pattern of impaired activation under conditions of oxidative stress, we investigated the potential contribution of an oxidant dependent suppressive pathway on direct Treg mediated suppression of Teff in vitro using the antioxidant n-acetylcysteine (NAC) to block reactive oxidants. Methods: Tregs and Teff were derived from the spleens of 2–4 month old C57BL/6 mice maintained in pathogen free conditions. T cell subsets were isolated using magnetic bead based techniques. Purity was assessed by flow cytometry. For suppression assays, Tregs were co-cultured with CFSE-labeled Teff at the indicated ratios and stimulated with anti-CD3/CD28 coated beads for 3 days. Proliferation was measured by flow cytometric evaluation of CFSE dilutional staining. Suppression was calculated by comparing proliferation of Teff cultured alone to those co-cultured with Tregs (% suppression= 1 − Tregs:Teff/Teff alone). In other experiments, CFSE-labeled Teff were cultured alone in the indicated conditions with TGF-beta +/− NAC and proliferation was assessed by CFSE staining. Intracellular reactive oxygen species (ROS) were measured by flow cytometry using the redox sensitive cell permeable indicator dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (DCFDA). Results: The presence of NAC prevented Treg suppression of Teff in a dose dependent fashion at Treg:Teff ratios of 1:2, 1:1 and 2:1 (see figure). Suppression was significantly decreased at 0.1mM NAC (p<0.05 at 1:1; p<0.01 at 2:1) and essentially absent at 1mM NAC. Proliferation of Teff was markedly higher in the setting of 1mM NAC compared to conditions without NAC, even during co-culture with Tregs at a 2:1 ratio of Tregs:Teff. Treg suppression of Teff proliferation was dependent on TGF-beta as neutralizing antibodies reversed the effect (p<0.001). The presence of NAC was sufficient to overcome the suppressive effects of exogenous TGF-beta on CD3/CD28 stimulated Teff proliferation. Treatment of CD3/CD28 stimulated Teff with TGFbeta resulted in a significant dose dependent increase in the levels of intracellular ROS (p<0.0001 at 10ng/mL TGF-beta) that inversely correlated with the degree of proliferation. Conclusion: NAC blocks Treg mediated TGF-beta dependent suppression in vitro. This suggests that TGF-beta may function to suppress proliferation of Teff via a ROS dependent mechanism and raises the possibility that targeted delivery of antioxidants may have clinical utility for modulating the effects of Tregs in vivo. Disclosures: No relevant conflicts of interest to declare.

Iron Overload Effects On Immune System Through The Cytokine Secretion By Macrophage

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1047-1047 ◽  
Author(s):  
Keiko Matsui ◽  
Sachiko Ezoe ◽  
Takafumi Yokota ◽  
Tomohiko Ishibashi ◽  
Kenji Oritani ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Iron Overload ◽  
Th17 Cell ◽  
Th17 Cells ◽  
Iron Dextran ◽  
Immune Systems ◽  
Treg Population

Abstract Iron is essential for almost all organisms. However, free iron can be cytotoxic at high concentration and iron excess can have adverse effects on variety of cells, tissue, and organ functions. In immune systems, many reports have shown the effects of iron, some of which are complex and controversial. Iron deficiency has been reported to be associated with increased susceptibility to infection, but iron overload caused by dietary excess abnormal hemolysis or inherited disorders is also associated with heightened susceptibility to infections. On the other hand, elevated ferritin levels, primarily related to RBC transfusions, have been reported to increase the risk of acute and chronic GVHD in patients received hematopoietic cell transplantation. Although this iron-related risk of GVHD may reflect the organ damage, such as liver, kidney, and pancreas, it also may be caused by imbalance in immune systems. To exaggerate the effects of iron overload on immune system, we established iron-loaded models in mice. First, as acute iron-loaded model, 10mg of iron dextran, which is even equal to 200U of RCC transfusion in human (define as 200U of iron dextran), was intraperitoneally injected into mice once a day for 18 days. In peripheral blood, T cell and B cell populations were decreased but monocyte/macrophage and neutrophil populations were increased as compared in control mice administered with dextran only. And notably, regulatory T cell (CD4+Foxp3+; Treg) population was significantly reduced in iron loaded mice than control mice (1.40% vs 0.58%). Next, as chronic iron-loaded model, 2U of iron dextran was injected once a week 45 times. Although significant difference was not observed in Treg population in PB, that in spleen was significantly high in iron-loaded mice as compared with that in control mice (3.2% vs 1.7%, p<0.05). From these data, we suspected that the excess of iron reduces Treg population in some organs, thereby effects on immune function. To clarify the mechanism of Treg-reduction by iron overload, we established an intermediate iron-loaded model, in which 50U of iron dextran was injected into mice once a week for 10 weeks, and following experiments were performed in this model. As previous examinations, Treg population in spleen was reduced in iron-loaded mice (0.86% vs 0.46%). T helper 17 (Th17) cells are recently described lymphocyte subset with common precursors with Treg but with opposing actions to Treg. The difference of Th17 cells could not be detected in spleen because of its too small population, the population of Th17 was increased in small intestine of iron-loaded mice (0.85% vs 1.51%). Treg and Th17 cells can be differentiated from naïve Th cells (CD4+CD62L+) with stimulation of specific cytokines (Treg; TGFβ, IFNγ, and IL4, Th17; TGFβ, IL-6, and IL-1β). We analyzed the direct effects of iron on Treg and Th17 cells during the in vitro differentiation from naïve T cells. Both in Treg and Th17 cell inductions, we could not detect significant differences between cells supplemented with iron dextran and those with dextran only. We also exaggerated the reactive oxide spesies (ROS) accumulated in induced T cell using RedCC1 staining. During the induction of Treg and Th17, although H2O2 supplement increased the ROS accumulation dose dependently, iron dextran-addition did not change the amount of ROS. Next, we analyzed the serum concentrations of cytokines in iron-loaded mice both in acute and intermediate models. IL-1β, and IL-23 levels were elevated in iron-loaded mice. These data suggested that iron overload reduces Treg cell population and increases Th17 cell population not directly, but through the cytokine secretion from environmental cells. So we evaluated the induction rates of Treg and Th17 cells from naïve T cells under co-culture with monocyte/macrophage. In this condition, Treg induction rate was significantly lower in iron dextran-supplemented cells (48.1% vs 35.3%), and Th17 induction was increased (8.4% vs 18.0%). Furthermore, mRNA expressions of IL-23 and IL-β in macrophages were increased with iron dextran-supplement in in vitro culture. These data suggest that iron overload changes the balance of Treg and Th17 cells through the proliferation and activation of macrophages, and thereby effects on the immunological condition of some disease, such as GVHD and autoimmune. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Endothelial Cell Dysfunction Is Involved in the Progression of Myelodysplastic Syndromes

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3668-3668
Author(s):  
Tong Xing ◽  
Zhong-Shi Lyu ◽  
Cai-Wen Duan ◽  
Qi Wen ◽  
Hong-Yan Zhao ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
Myelodysplastic Syndromes ◽  
T Cell Subsets ◽  
Leukemia Cells ◽  
Rna Seq

Abstract Background: Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis, refractory anemia, and a tendency to transform to acute myeloid leukemia (AML). Ineffective hematopoiesis progression and immune deregulation are dominating pathophysiological process of MDS. Emerging evidences showed the role of bone marrow (BM) microenvironment in MDS. In MDS murine model, integral BM microenvironment contributes to inferior hematopoietic function and disease progression. As an important component of BM microenvironment, the relationship between endothelial cells (ECs) and MDS progression remains largely unknown. Although ECs from MDS patients have been identified to have decreased supporting ability to normal hematopoietic stem cells (HSCs), the supporting ability of ECs in different clinical stages of MDS remains to be elucidated. In addition, the role of BM ECs from MDS patients in supporting leukemia cells and their immunomodulatory ability remains unclear. Aims: To determine the number and functions of BM ECs in different subtypes of MDS patients. Moreover, to explore the correlation between BM ECs and MDS progression, which may represent a potential therapeutic target for MDS patients. Methods: In the prospective cohort study, patients with multilineage dysplasia (MDS-MLD, N=15), MDS with excess blasts (MDS-EB, N=15), or AML(N=15) and healthy donors (HD, N=15) were enrolled. BM ECs were analyzed in HD and patients by flow cytometry and in situ histological analyses. The functions of BM ECs were analyzed by migration, angiogenesis capacities, levels of apoptosis and reactive oxygen species (ROS). To evaluate the supporting abilities of BM ECs on HSCs, leukemia cells and T cells, in vitro co-culture strategies were used. The levels of apoptosis, ROS and colony-forming unit-plating (CFU) efficiency of CD34+ and HL-60 cells were investigated. T cell subsets were analyzed by flow cytometry as previously reported. To further investigate the underlying mechanism of dysfunctional ECs, RNA sequencing (RNA-Seq) analyses and real time-PCR (qRT-PCR) were performed in BM ECs from HD and MDS patients with different subtypes. Results: In the current study, gradually increased BM ECs were observed from MDS-MLD, MDS-EB to AML patients. Furthermore, dysfunctional BM ECs were found with MDS progression, characterized by increased levels of migration, angiogenesis capacities, apoptosis and ROS. More importantly, BM ECs from MDS patients exhibited decreased supporting ability of HSCs whereas increased supporting ability of leukemia cells in vitro with MDS progression. After coculture with ECs, levels of apoptosis and ROS in CD34+ cells were increased whereas their CFU efficiency reduced. On the other hand, levels of apoptosis and ROS of HL-60 cells were decreased. The proliferation capacity and leukemia CFU efficiency of HL-60 cells after co-cultured with ECs were enhanced with MDS progression. Furthermore, following coculture with BM ECs, deregulated differentiation was demonstrated in T cell subsets, characterized by elevating proportion of Th2 and Treg and decreasing proportion of Th1 and Th17 with MDS progression. RNA-Seq showed that the expression profile of BM ECs from MDS-EB was closer to MDS-MLD, whereas that of MDS-EB was closer to AML. Different gene expression profiles indicated the expression of hematopoiesis and immune related genes increased in BM ECs with MDS progression. Mechanistically, the mRNA levels of CX CL12, SCF and NFKB of ECs were increased with MDS progression. Summary/Conclusion: In summary, the number of BM ECs gradually increased, BM EC dysfunction more and more severe, and the supporting abilities of BM ECs on HSCs decreased, whereas on leukemia cells increased with MDS progression. Moreover, ECs regulated the differentiation of T cells into immune tolerant cells with MDS progression. Although further validation is required, these findings indicated that the improvement of BM ECs may represent a potential therapeutic approach for MDS patients. Keywords: Myelodysplastic syndromes, endothelial cells, disease progression, ineffective hematopoiesis, immune deregulation Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional characteristics of Th1, Th17, and ex-Th17 cells in EAE revealed by intravital two-photon microscopy

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Julia Loos ◽  
Samantha Schmaul ◽  
Theresa Marie Noll ◽  
Magdalena Paterka ◽  
Miriam Schillner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Th17 Cells ◽  
Laser Scanning Microscopy ◽  
T Cell Subsets ◽  
Th1 Cells ◽  
Two Photon ◽  
Functional Dynamics

Abstract Background T helper (Th) 17 cells are a highly plastic subset of T cells, which in the context of neuroinflammation, are able to acquire pathogenic features originally attributed to Th1 cells (resulting in so called ex-Th17 cells). Thus, a strict separation between the two T cell subsets in the context of experimental autoimmune encephalomyelitis (EAE) is difficult. High variability in culture and EAE induction protocols contributed to previous conflicting results concerning the differential contribution of Th1 and Th17 cells in EAE. Here, we systematically evaluate the role of different T cell differentiation and transfer protocols for EAE disease development and investigate the functional dynamics of encephalitogenic T cells directly within the inflamed central nervous system (CNS) tissue. Methods We compiled the currently used EAE induction protocols reported in literature and investigated the influence of the different Th1 and Th17 differentiation protocols as well as EAE induction protocols on the EAE disease course. Moreover, we assessed the cytokine profile and functional dynamics of both encephalitogenic Th1 and Th17 cells in the inflamed CNS using flow cytometry and intravital two-photon laser scanning microscopy. Lastly, we used astrocyte culture and adoptive transfer EAE to evaluate the impact of Th1 and Th17 cells on astrocyte adhesion molecule expression in vitro and in vivo. Results We show that EAE courses are highly dependent on in vitro differentiation and transfer protocols. Moreover, using genetically encoded reporter mice (B6.IL17A-EGFP.acRFP x 2d2/2d2.RFP), we show that the motility of interferon (IFN)γ-producing ex-Th17 cells more closely resembles Th1 cells than Th17 cells in transfer EAE. Mechanistically, IFNγ-producing Th1 cells selectively induce the expression of cellular adhesion molecules I-CAM1 while Th1 as well as ex-Th17 induce V-CAM1 on astrocytes. Conclusions The behavior of ex-Th17 cells in EAE lesions in vivo resembles Th1 rather than Th17 cells, underlining that their change in cytokine production is associated with functional phenotype alterations of these cells.

scholarly journals Quantitative analysis and genome-scale modeling of human CD4+ T-cell differentiation reveals subset-specific regulation of glycosphingolipid pathways

2021 ◽  
Author(s):  
Partho Sen ◽  
Syed Bilal Ahmad Andrabi ◽  
Tanja Buchacher ◽  
Mohd Moin Khan ◽  
Ubaid Ullah ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Th17 Cells ◽  
Functional Differentiation ◽  
Metabolic Reprogramming ◽  
Gene Knockdown ◽  
T Cell Subsets ◽  
Genome Scale

ABSTRACTT-cells are sentinels of adaptive cell-mediated immune responses. T-cell activation, proliferation and differentiation involves metabolic reprogramming involving the interplay of genes, proteins and metabolites. Here, we aim to understand the metabolic pathways involved in the activation and functional differentiation of human CD4+ T-cell subsets (Th1, Th2, Th17 and iTregs). We combined genome-scale metabolic modeling, gene expression data, targeted and non-targeted lipidomics experiments, together with in vitro gene knockdown experiments and showed that human CD4+ T-cells undergo specific metabolic changes during activation and functional differentiation. In addition, we identified and confirmed the importance of ceramide and glycosphingolipid synthesis pathways in Th17 differentiation and effector functions. Finally, through in vitro gene knockdown experiments, we substantiated the requirement of serine palmitoyl transferase (SPT), a de novo sphingolipid pathway in the expression of proinflammatory cytokine (IL17A and IL17F) by Th17 cells. Our findings may provide a comprehensive resource for identifying CD4+ T-cell-specific targets for their selective manipulation under disease conditions, particularly, diseases characterized by an imbalance of Treg / Th17 cells. Our data also suggest a role for elevated levels of ceramides in conditions comorbid with these diseases, e.g., obesity and insulin resistance.

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