THE EFFECT OF GLUTAMATE ON THE MIGRATION OF T CELLS FROM HEALTHY DONORS AND PATIENTS WITH MULTIPLE SCLEROSIS IN VITRO

Aim of study. To study chemotactic properties of glutamate and glutamate receptor agonists on T cells migration from healthy donors and patients with multiple sclerosis (MS) in vitro. Materials and methods. T cell migration of 15 patients with MS and 15 healthy donors was studied in vitro using transwells. Lymphocytes were activated with PMA (10 ng/mL). T cells were added to transwells with fibronectin (10 μg/mL) pretreated membrane. The lower chamber contained glutamate or AMPA or NMDA (100 μM for each) in complete RPMI medium. Migrated cells were collected and stained with antibodies to CD3-marker for subsequent analysis by cytofluorimetry. Results and conclusion. In presence of glutamate, there is a tendency to a decrease in migration activity in both groups of donors. T-cell chemotaxis of healthy donors, but not MS patients, decreased in concentration gradient of NMDA. The activation of lymphocytes with PMA leads to a decrease in the number of migrated cells by an average of 17% (p < 0.01). In MS patients there is a tendency to an increase in chemotaxis of activated cells in concentration gradient of glutamate, and a decrease with AMPA. Thus, glutamate and glutamate receptors agonists do not possess pronounced chemotactic properties, but rather enhance T-cell migration through synthesis of adhesion molecules on the surface of lymphocytes and endothelium.

Download Full-text

Vitamin D/CD46 Crosstalk in Human T Cells in Multiple Sclerosis

Frontiers in Immunology ◽

10.3389/fimmu.2020.598727 ◽

2020 ◽

Vol 11 ◽

Author(s):

Justin Killick ◽

Joanne Hay ◽

Elena Morandi ◽

Sonja Vermeren ◽

Saniya Kari ◽

...

Keyword(s):

Multiple Sclerosis ◽

Vitamin D ◽

T Cells ◽

T Cell ◽

Chronic Inflammatory Disease ◽

Vitamin D Supplementation ◽

Type I ◽

T Cell Migration ◽

The Impact

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), in which T-cell migration into the CNS is key for pathogenesis. Patients with MS exhibit impaired regulatory T cell populations, and both Foxp3+ Tregs and type I regulatory T cells (Tr1) are dysfunctional. MS is a multifactorial disease and vitamin D deficiency is associated with disease. Herein, we examined the impact of 1,25(OH)2D3 on CD4+ T cells coactivated by either CD28 to induce polyclonal activation or by the complement regulator CD46 to promote Tr1 differentiation. Addition of 1,25(OH)2D3 led to a differential expression of adhesion molecules on CD28- and CD46-costimulated T cells isolated from both healthy donors or from patients with MS. 1,25(OH)2D3 favored Tr1 motility though a Vitamin D-CD46 crosstalk highlighted by increased VDR expression as well as increased CYP24A1 and miR-9 in CD46-costimulated T cells. Furthermore, analysis of CD46 expression on T cells from a cohort of patients with MS supplemented by vitamin D showed a negative correlation with the levels of circulating vitamin D. Moreover, t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis allowed the visualization and identification of clusters increased by vitamin D supplementation, but not by placebo, that exhibited similar adhesion phenotype to what was observed in vitro. Overall, our data show a crosstalk between vitamin D and CD46 that allows a preferential effect of Vitamin D on Tr1 cells, providing novel key insights into the role of an important modifiable environmental factor in MS.

Download Full-text

Form and pattern of MUC1 expression on T cells activated in vivo or in vitro suggests a function in T-cell migration

Immunology ◽

10.1046/j.1365-2567.2003.01562.x ◽

2003 ◽

Vol 108 (1) ◽

pp. 32-41 ◽

Cited By ~ 46

Author(s):

Isabel Correa ◽

Tim Plunkett ◽

Anda Vlad ◽

Arron Mungul ◽

Jessica Candelora-Kettel ◽

...

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Muc1 Expression ◽

T Cell Migration

Download Full-text

Modulation of Tetraspanin 32 (TSPAN32) Expression in T Cell-Mediated Immune Responses and in Multiple Sclerosis

International Journal of Molecular Sciences ◽

10.3390/ijms20184323 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4323 ◽

Cited By ~ 10

Author(s):

Salvo Danilo Lombardo ◽

Emanuela Mazzon ◽

Maria Sofia Basile ◽

Giorgia Campo ◽

Federica Corsico ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

T Cell ◽

Immune Responses ◽

Cancer Metastasis ◽

Treg Cells ◽

T Helper ◽

Autoimmune Encephalomyelitis ◽

Healthy Donors

Tetraspanins are a conserved family of proteins involved in a number of biological processes including, cell–cell interactions, fertility, cancer metastasis and immune responses. It has previously been shown that TSPAN32 knockout mice have normal hemopoiesis and B-cell responses, but hyperproliferative T cells. Here, we show that TSPAN32 is expressed at higher levels in the lymphoid lineage as compared to myeloid cells. In vitro activation of T helper cells via anti-CD3/CD28 is associated with a significant downregulation of TSPAN32. Interestingly, engagement of CD3 is sufficient to modulate TSPAN32 expression, and its effect is potentiated by costimulation with anti-CD28, but not anti-CTLA4, -ICOS nor -PD1. Accordingly, we measured the transcriptomic levels of TSPAN32 in polarized T cells under Th1 and Th2 conditions and TSPAN32 resulted significantly reduced as compared with unstimulated cells. On the other hand, in Treg cells, TSPAN32 underwent minor changes upon activation. The in vitro data were finally translated into the context of multiple sclerosis (MS). Encephalitogenic T cells from Myelin Oligodendrocyte Glycoprotein (MOG)-Induced Experimental Autoimmune Encephalomyelitis (EAE) mice showed significantly lower levels of TSPAN32 and increased levels of CD9, CD53, CD82 and CD151. Similarly, in vitro-activated circulating CD4 T cells from MS patients showed lower levels of TSPAN32 as compared with cells from healthy donors. Overall, these data suggest an immunoregulatory role for TSPAN32 in T helper immune response and may represent a target of future immunoregulatory therapies for T cell-mediated autoimmune diseases.

Download Full-text

MiR-155 Impacts T Cell Migration in Acute Graft-Versus-Host Disease (aGVHD)

Blood ◽

10.1182/blood.v126.23.3080.3080 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3080-3080 ◽

Cited By ~ 1

Author(s):

Nina C. Zitzer ◽

Patricia A. Taylor ◽

Apollinaire Ngankeu ◽

Yvonne A. Efebera ◽

Steven M. Devine ◽

...

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Chemokine Receptors ◽

Ccr5 Expression ◽

Post Transplant ◽

T Cell Migration ◽

T Cell Infiltration ◽

Donor T Cells

Abstract Introduction: We reported that microRNA-155 (miR-155) expression is upregulated in donor T cells during aGVHD and mice receiving miR-155 knock-out (KO) donor splenocytes do not exhibit lethal GVHD and have improved survival as compared to mice receiving wild type (WT) splenocytes.1 While we showed that miR-155 does not affect the allo-reactive proliferative potential of T cells, a significant decrease in the expression of the homing receptors CCR5, CXCR4, and S1P1 was found on miR-155-KO T cells, suggesting that the loss of miR-155 could impair the migration of donor T cells to aGVHD target organs resulting in less lethality. Here, we further investigate the impact of miR-155 expression in T cell migration. Materials and Methods: Lethally irradiated BALB/c or B6D2F1 recipients were infused with T cell depleted WT bone marrow (BM) cells (5x10^6) and GFP expressing miR-155 KO or GFP-B6 WT T cells (1x10^6). Recipients were sacrificed at day 7, 14 and 21 post-transplant, organs harvested and donor T cell infiltration evaluated via confocal microscopy. Transwell migration assays towards CCR5 ligands macrophage inflammatory protein-1a (MIP-1a) (100ng/mL) and RANTES (100ng/mL) was performed utilizing WT or miR-155-KO T cells activated using irradiated BALB/c splenocytes as allogeneic stimulators at a stimulator: responder ratio of 1:5. Lower chambers with medium only served as a control for spontaneous migration. CCR5 ligand-dependent migration was calculated according to the formula: Migration Index (MI) = number of cells CCR5 ligands / number of cells medium only. Results: On days 7, 14 and 21 post transplant, recipient mice were sacrificed, and tissues harvested in order to study the kinetics of miR-155 KO T cell migration following allogeneic hematopoietic stem cell transplant. There was a dramatic decrease in T cell infiltration of peripheral organs (PeyerÕs patches, liver, lung and skin) in recipients of miR-155-KO T cells as compared to WT T cells as evidenced by confocal microscopy of GFP labeled donor cells, Figure 1. We reasoned that these effects could be due to the modulation of CCR5 and other chemokine receptors by miR-155. There was a significant decrease in CCR5 mRNA and protein expression in miR-155-KO versus WT donor T cells obtained from recipient mice at the time of aGVHD. To demonstrate the functional significance of decreased CCR5 expression in miR-155 KO donor T cells, we performed in vitro transwell migration assays to CCR5 ligands RANTES and MIP-1a. To our knowledge, we are the first to show that allo-activated miR-155 KO T cells show significantly reduced migration towards CCR5 ligands, as demonstrated by the average MI of 1.08, when compared to the average MI of WT T cells of 4.79, p=0.004, Figure 2. There were lower percentages of CCR5 positive T cells and decreased mean fluorescent intensity in the miR-155 KO T cells after allogeneic stimulation when compared to WT T cells, both in the CD4+ and CD8+ populations, confirming lower CCR5 expression in miR-155 KO T cells after in vitro allogeneic stimulation. To further elucidate the mechanism of miR-155 mediated modulation of CCR5 expression, we focused on long non-coding RNA (lncRNA) LincR-Ccr2-5′AS located in the vicinity of several chemokine receptor encoding genes including CCR1, CCR2 and CCR5, known to be important for migration of Th2 cells. We found that LincR-Ccr2-5′AS has 3 potential miR-155 binding sites and so set out to determine if miR-155 negatively regulates the expression of this lncRNA, thereby influencing chemokine receptor expression as well as T cell migration. We isolated T cells from B6D2F1 recipients 21 days post-transplant, and showed a significant decrease in CCR5 mRNA expression in miR-155 KO versus WT donor T cells but no significant difference in the levels of LincR-Ccr2-5′AS. However, this result does not exclude the possibility that miR-155 might influence the activity rather than the levels of LincR-Ccr2-5′AS, which we hope to determine in future experiments. Conclusion: Our data suggest that miR-155 may exert its modulating effects in aGVHD by affecting T cell migration. Experiments are currently underway to determine the role of miR-155 in modulating T cell migration through other chemokine receptors such as CXCR4, as well as S1P1 and ATP receptor P2X7R. Reference 1. Ranganathan P, Heaphy CE, Costinean S, et al. Regulation of acute graft-versus-host disease by microRNA-155. Blood. 2012 May 17;119(20):4786-97. Disclosures No relevant conflicts of interest to declare.

Download Full-text

CrkL is required for donor T cell migration to GvHD Target organs

10.1101/854042 ◽

2019 ◽

Author(s):

Nathan H. Roy ◽

Mahinbanu Mammadli ◽

Janis K. Burkhardt ◽

Mobin Karimi

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Adaptor Protein ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Peripheral Tissues ◽

T Cell Migration ◽

Related Family

ABSTRACTThe success of cancer therapies based on allogeneic hematopoietic stem cell transplant relies on the ability to separate graft-versus-host disease (GvHD) from graft-versus-tumor (GVT) responses. Controlling donor T cell migration into peripheral tissues is a viable option to limit unwanted tissue damage, but a lack of specific targets limits progress on this front. Here, we show that the adaptor protein CrkL, but not the closely related family members CrkI or CrkII, is a crucial regulator of T cell migration. In vitro, CrkL-deficient T cells fail to polymerize actin in response to the integrin ligand ICAM-1, resulting in defective migration. Using a mouse model of GvHD/GVT, we found that while CrkL-deficient T cells can efficiently eliminate hematopoietic tumors they are unable to migrate into inflamed organs, such as the liver and small intestine, and thus do not cause GvHD. These results suggest a specific role for CrkL in trafficking to peripheral organs but not the lymphatic system. In line with this, we found that although CrkL-deficient T cells could clear hematopoietic tumors, they failed to clear the same tumor growing subcutaneously, highlighting the role of CrkL in controlling T cell migration into peripheral tissues. Our results define a unique role for CrkL in controlling T cell migration, and suggest that CrkL function could be therapeutically targeted to enhance the efficacy of immunotherapies involving allogeneic donor cells.

Download Full-text

PLCβ Is Critical for T-Cell Chemotaxis In Vivo.

Blood ◽

10.1182/blood.v104.11.2650.2650 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2650-2650

Author(s):

Tami L. Bach ◽

Qing-Min Chen ◽

Martha S. Jordan ◽

John K. Choi ◽

Dianqing Wu ◽

...

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Chronic Inflammation ◽

Critical Role ◽

Neutrophil Chemotaxis ◽

Wild Type ◽

T Cell Migration

Abstract Chemokines acting through G-protein coupled receptors play an essential role in both the immune and inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) are two distinct signaling molecules that have been proposed as potential candidates in the regulation of this process. Studies with knockout mice have demonstrated a critical role for PI3Kγ, but not PLCβ, in Gαi-coupled receptor-mediated neutrophil chemotaxis. We compared the chemotactic response of peripheral T-cells derived from wild type mice with mice containing loss-of-function mutations of either PI3Kγ, or both of the two predominant lymphocyte PLCβ isoforms (PLCβ2 and PLCβ3). In contrast to neutrophils, loss of PI3Kγ did not significantly impair T-cell migration in vitro, although PI3K pharmacologic inhibitor experiments suggest that another isoform of this enzyme might contribute to T-cell migration. However, loss of PLCβ2β3 decreased chemokine-stimulated T-cell migration in vitro. Chelation of intracellular calcium by BAPTA-AM and Quin-2 AM decreased the chemotactic response of wild type lymphocytes, but pharmacologic inhibition of PKC isoforms by GF109203x did not impair T-cell migration. This suggests that the T-cell migration defect seen in the PLCβ2β3-null T-cells may be due to an impaired ability to increase the cytoplasmic calcium concentration, while there appears to be little requirement for PKC activity. Indeed, SDF-1α-induced calcium efflux was not detected in the PLCβ2β3-null lymphocytes. Compared to fluorescently labeled wild type T-cells, labeled PLCβ2β3 knockout T-cells migrated less efficiently into secondary lymphoid organs of recipient mice. This demonstrates that PLCβ is also required for migration in vivo. PLCβ2β3-null mice develop spontaneous skin ulcers starting around 3 months of age. Histological examination of the lesions revealed a dense inflammatory infiltrate composed of neutrophils, macrophages, and plasma cells, consistent with acute and chronic inflammation. Remarkably, lymphocytes, typical of chronic inflammation, were rare to absent by histology and by paraffin immunohistochemistry for CD3, also consistent with an in vivo migratory defect of T-cells. These results show that phospholipid second messengers generated by PLCβ and isoforms of PI3K, other than PI3Kγ, play a critical role in lymphocyte chemotaxis. Collectively, our data demonstrate that although PLCβ-mediated signaling plays no role in neutrophil chemotaxis, it makes a substantial contribution to this process within T-lymphocytes.

Download Full-text

T Cell Lymphopenia in Rhoh−/− Mice Results from Combined Defects of T Cell Migration and IL-7-Regulated Naïve T Cell Homeostasis.

Blood ◽

10.1182/blood.v108.11.872.872 ◽

2006 ◽

Vol 108 (11) ◽

pp. 872-872

Author(s):

ShuShun Li ◽

David A. Hildeman ◽

H. Leighton Grimes ◽

David A. Williams ◽

Yi Gu

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Cell Survival ◽

T Cell Homeostasis ◽

Cell Homeostasis ◽

T Cell Migration ◽

T Cell Lymphopenia ◽

T Cell Survival

Abstract The Rho family GTPases are increasingly implicated for their important roles in T cell development and function. We have found that RhoH, a hematopoietic-specific member of this family is essential for thymocyte development (Gu et al, Nat Immunol, in press). Further, Rhoh−/− mice showed T cell lymphopenia. In particular, naïve T cells were reduced in secondary lymphoid organs and blood both in unchallenged and LCMV-challenged Rhoh−/− mice compared to WT controls. These findings suggest a possible defect in T cell emigration from thymus and peripheral T cell homeostasis in Rhoh−/− mice. To study the role(s) of RhoH in T cell migration and homeostasis, purified splenic T cells were adoptively transferred into common γ−/−; Rag2−/− mice. T cells were then recovered from spleens 3d and 8d post-transplantation. The recovery of Rhoh−/− T cells was decreased by 60% compared with WT cells. In vitro migration of Rhoh−/− T cells towards SDF-1α, a homeostatic chemokine for T cells, in a transwell migration assay was significantly reduced compared to WT cells. Flow analysis showed decreased number of CXCR4 expressing and reduced expression levels of CXCR4 on Rhoh−/− T cells. Furthermore, apoptotic T cells were increased twofold in Rhoh−/− mice compared to controls. CFSE staining of adoptively transferred T cells demonstrated a comparable proliferation rate between Rhoh−/− and WT cells in the recipient mice. Our data suggest that RhoH plays an important role in T cell homeostasis via regulating cell survival and SDF-1α-mediated migration. To further investigate how RhoH regulates T cell survival, we focused on IL-7, an essential factor for prolonged survival of naïve T cells. One way IL-7 exerts its effect is through upregulating the Bcl-2 family of antiapoptotic proteins. Our data showed that in vitro survival of Rhoh−/− T cells in response to IL-7 was impaired, and expression of IL-7Rα and Bcl-2 were both decreased in Rhoh−/− T cells. To further study a potential role of RhoH in regulation of IL-7R expression, FACsvantage sorted naïve T cells (CD4+CD44low, or CD8+CD44low) were cytokine starved overnight, then cultured with or without the addition of IL-7 for 6 hrs, and analyzed for IL-7Rα expression by flow cytometry. In WT cells, during cytokine starvation CD4 and a subset of CD8 naïve cells upregulated IL-7Rα expression, but this upregulation was reduced followed IL-7 stimulation. In contrast, Rhoh−/− T cells failed to show either up- or subsequent down-regulation of IL-7Rα in response to cytokine starvation and IL-7 exposure. These data may indicate a role for RhoH in regulating IL-7R expression in naïve CD4 T cells. Taken together, these findings suggest that RhoH is required for IL-7-mediated T cell survival and SDF-1α-mediated homing and/or emigration from thymus. Thus, deficiency of naïve T cells in Rhoh−/− mice likely results from combined defects in T cell migration and homeostasis.

Download Full-text

Regulatory T cells reduce endothelial neutral sphingomyelinase 2 to prevent T cell migration into tumors

European Journal of Immunology ◽

10.1002/eji.202149208 ◽

2021 ◽

Author(s):

Paulina Akeus ◽

Louis Szeponik ◽

Veronica Langenes ◽

Viktoria Karlsson ◽

Patrik Sundström ◽

...

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Regulatory T Cells ◽

Neutral Sphingomyelinase ◽

T Cell Migration

Download Full-text

Novel self-amplificatory loop between T cells and tenocytes as a driver of chronicity in tendon disease

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-219335 ◽

2021 ◽

pp. annrheumdis-2020-219335

Author(s):

Emma Garcia-Melchor ◽

Giacomo Cafaro ◽

Lucy MacDonald ◽

Lindsay A N Crowe ◽

Shatakshi Sood ◽

...

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Inflammatory Cytokines ◽

T Cell Activation ◽

Cell Activation ◽

Conditioned Media ◽

Tissue Remodelling ◽

Activated T Cells ◽

T Cell Migration

ObjectivesIncreasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation.MethodsT cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes.ResultsSignificant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio.ConclusionsInteraction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders.

Download Full-text

Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

Clinical and Developmental Immunology ◽

10.1155/2008/384982 ◽

2008 ◽

Vol 2008 ◽

pp. 1-8 ◽

Cited By ~ 44

Author(s):

Shumei Man ◽

Eroboghene E. Ubogu ◽

Katherine A. Williams ◽

Barbara Tucky ◽

Melissa K. Callahan ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

T Cell ◽

Human Brain ◽

Umbilical Vein ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

T Cell Migration ◽

Umbilical Vein Endothelial Cells

Endothelial cells that functionally express blood brain barrier (BBB) properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs) and human umbilical vein endothelial cells (HUVECs). With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER) and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

Download Full-text