While soluble fractalkine induces changes in L-selectin surface expression, that of ICAM-1, VLA-4, LFA-1, alpha-4, and beta-integrin together with memory phenotype are unchanged

While soluble fractalkine induces changes in L-selectin surface expression, that of ICAM-1, VLA-4, LFA-1, alpha-4, and beta-integrin together with memory phenotype are unchanged. the accumulation of memory CD8+ T cells in the omentum of EAC patients. Our data show that fractalkine is significantly enriched in the omentum of EAC patients and drives migration of T cells derived from EAC patient blood. Furthermore, CX3CR1 is endocytosed specifically by CD8+ T cells upon encountering fractalkine, which is consistent with the significantly diminished frequencies of CX3CR1INT and CX3CR1HI CD8+ T cells in the fractalkine-rich environment of omentum in EAC, relative to matched blood. Fractalkine-mediated endocytosis of CX3CR1 by CD8+ T cells is sustained and is followed by enhanced surface expression of L-selectin (CD62L). These novel data align with our findings that circulating CX3CR1NEG CD8+ T cells express higher levels of L-selectin than CX3CR1INT CD8+ T cells. This is consistent with previous reports and implicates fractalkine in the conversion of CX3CR1INT CD8+ T cells to a CX3CR1NEG phenotype characterized by alterations in the migratory capacity of these T cells. For the first time, these findings identify fractalkine as a driver of T cell migration to the omentum in EAC and indicate that CD8+ T cells undergo sequenced fractalkine-mediated alterations in CX3CR1 and L-selectin expression. These data implicate fractalkine as more than a chemotactic cytokine in obesity-associated meta-inflammation and reveal a role for this chemokine in the maintenance of the CX3CR1NEG CD8+ T cell populations. coomassie blue staining (10% gel, 20?g protein per sample). CD8+ T cells from three control subjects were isolated from PBMC using the EasySep? Human CD8+ T Cell Isolation Kit (Stemcell Technologies) and subsequently seeded in RPMI media at 1??106 cells/ml and treated with 30?ng/ml of fractalkine for 24 and 48?h. Cell supernatant was collected after 24 and 48?h and the Human CX3CR1 ELISA Kit (ELISA Genie) was used to compare secreted CX3CR1 in the untreated and fractalkine-treated cells. Assessing Integrin and Adhesion Molecule Expression Together With Memory Phenotype of CD8+ T Cells Following Fractalkine Treatment To examine the effects of fractalkine on CX3CR1 expression by CD8+ T cells, PBMC from six EAC patients were treated Bay 60-7550 with M199 media alone or M199 media supplemented with 30?ng/ml of recombinant fractalkine for 24?h and subsequently analyzed for VLA-4, LFA-1, alpha4 integrin, beta7 integrin, ICAM-1, L-selectin, CD45RA, and CD27 surface expression using flow cytometry, as described above. Statistical Analyses Statistical analysis was carried out using Prism GraphPad Version 5.0. Differences between groups were assessed using two-tailed paired, Wilcoxon sign-rank test, unpaired non-parametric MannCWhitney tests, and one-way ANOVA with Tukey analysis where appropriate. Significant associations between fractalkine, CX3CR1, and clinical parameters were investigated using Spearmans rank-order correlation test. Values of <0.05 were considered to be significant. Results Significantly High Levels of Soluble Fractalkine in the Omentum of EAC Patients Can Drive Migration of EAC Patient-Derived T Cells Secreted fractalkine was quantified by MSD V-Plex ELISA in the matched serum and omental adipose tissue conditioned media (ACM) of 19 EAC patients revealing that levels of this chemokine were significantly higher in ACM (mean: 23.66?ng/ml) compared to serum (mean: 10.56?ng/ml) (tests and one-way ANOVA with Tukey analysis. Table 2 Correlations of CX3CL1 levels and frequencies of CX3CR1NEG expressing T cells with waist circumference, visceral fat area (VFA), and body mass index. tests. CX3CR1 Expression by Peripheral Blood but Not Omental CD8+ T Cells Is Significantly Diminished Following Treatment With Recombinant Fractalkine To ascertain why enrichments of CX3CR1+ CD4+ T cells were detected in the omentum, while highest frequencies of CX3CR1+ CD8+ T cells were detected in the circulation, we Bay 60-7550 assessed whether CX3CR1+ CD8+ T cells convert to CX3CR1NEG CD8+ T cells upon encountering their ligand, which is secreted in abundance in the omental microenvironment. Blood-derived T cells from 17 EAC patients were treated with M199 media or recombinant fractalkine for 2?h to simulate the effects of the high fractalkine levels in the omental microenvironment. Flow cytometric analysis revealed that surface expression of CX3CR1 was significantly decreased on peripheral blood CD8+ but not CD4+ T cells or omental CD8+ T cells following 2?h treatment with recombinant fractalkine (untreated versus treated CD8+ T cells: 52.2 Mouse monoclonal to CD3.4AT3 reacts with CD3, a 20-26 kDa molecule, which is expressed on all mature T lymphocytes (approximately 60-80% of normal human peripheral blood lymphocytes), NK-T cells and some thymocytes. CD3 associated with the T-cell receptor a/b or g/d dimer also plays a role in T-cell activation and signal transduction during antigen recognition versus 4.238, tests. Open in a separate window Figure 4 CX3CR1 is endocytosed following fractalkine treatment and is not subsequently recycled to the surface of CD8+ T cells or secreted but, intracellular accumulations of the protein are detectable. (A) Frequencies of CX3CR1+ cells, as a percentage of CD8+ T cells following treatment with M199 media alone (NT), 30?ng/ml recombinant fractalkine alone (treatment alone), 30?ng/ml recombinant fractalkine at 4C and 30?ng/ml recombinant fractalkine plus Bay 60-7550 80?M Dynasore (tests. Increased L-Selectin Surface Expression Follows Fractalkine-Mediated CX3CR1 Endocytosis on Bay 60-7550 CD8+ T.