Plates were incubated at 37 for 1 hr, after which, filter inserts were washed to remove non-adherent cells. of ZAP-70 after CXCR3 activation is usually a Lck-dependent process. Finally, stimulating CXCR3-expressing Jurkat T cells and normal T cells expressing CXCR3 through the T-cell receptor attenuated CXCR3-induced tyrosine phosphorylation and CXCR3-mediated T-cell Z-DEVD-FMK migration, indicating the occurrence of cross-talk between T-cell receptor and CXCR3-signalling pathways. These results shed light on the mechanisms of CXCR3 signalling. Such information could be useful when designing therapeutic strategies to regulate T-cell function. 005; 30 min MCF 579 130 versus 639 170, 005. CXCL11 (treated versus untreated): 0 min, MCF 4935 145 versus 635 195, 005; 10 min, 376 2306 versus 608 110, = 001; 30 min MCF 370 95 versus 6215 65, 0005. Open in a separate windows Physique 9 Activation through the TCR attenuates CXCL10-induced and CXCL11-induced protein tyrosine phosphorylation. (a) JE6.1/CXCR3 cells were incubated at 37 for 5 min in 0001% BSA/RPMI alone (lane 1) or with 0001% BSA/RPMI containing 3 g/ml anti-CD3 antibody (lane 2). After incubation, the cells were either lysed (a), or were washed and resuspended in 5% FCS/RPMI. The cells were then allowed to rest for 2 hr at 37 in 5% CO2. After resting, the cells were washed and incubated for 5 min on ice in 0001% BSA/RPMI alone (bCf: lane 1) or in 0001% BSA/RPMI made Z-DEVD-FMK up of 30 nm CXCL10 (bCd: lanes 2, 3) or 30 nm CXCL11 (e and f: lanes 2, 3). After incubation, the cells were relocated to a 37 water bath and incubated for 5 min. After incubation, the cells were lysed and the proteins in the whole cell lysates were separated by SDSCPAGE, transferred to membranes, and immunoblotted LEP with anti-phosphotyrosine mAb (b, e), with antibody specific for phosphotyrosine residue 319 in ZAP-70 (c and f, upper panels), or with the combination of antibodies specific for phosphotyrosine residues 191 and 171 in LAT (d and f, upper panels). After immunoblotting, membranes were stripped and reblotted with antibody against ZAP-70 (c and f, lower panels) or LAT (d and f, lower panels) to test for equal protein loading. Pre-CD3 indicates cells that were prestimulated with anti-CD3 antibody. Results The addition of CXCR3 ligands to normal human T cells and to Jurkat T cells designed to express CXCR3 induces protein tyrosine phosphorylation The molecular mechanisms underlying CXCR3 signalling are not clearly understood. Although CXCR3 is usually scantily expressed on quiescent T cells, it is strongly expressed on activated T cells.7,8,15,16,22,25C27 To induce the expression of CXCR3, T cells isolated from normal human volunteers were stimulated with PHA-L (500 ng/ml) in combination with human IL-2 (50 ng/ml) as described in the Material and methods. This approach reproducibly led to the stable expression of CXCR3 (Fig. 1). At least 90% of the cells expressed TCR (Fig. 1). Open Z-DEVD-FMK in a separate window Physique 1 CXCR3Cligand conversation in normal human T cells induces protein tyrosine phosphorylation. T cells were harvested from your blood of normal volunteers, stimulated with PHA (500 ng/ml) for 48 hr and then supplemented Z-DEVD-FMK with IL-2 (50 ng/ml). After 10 days in culture, FACS analysis of stimulated cells was performed by labelling cells with PE-conjugated anti-human-CXCR3 and FITC-conjugated anti-TCR- monoclonal antibody along with appropriate isotype controls for 30 min on ice. PI was used to exclude non-living cells from your FACS analysis. Panels symbolize per cent of cells positive for CXCR3 and TCR- as compared to isotype controls. Purified normal human T cells expressing CXCR3 were incubated on ice for 5 min Z-DEVD-FMK in 0001% BSA/RPMI alone (lane 1) or in 0001% BSA/RPMI made up of 100 nm of the CXCR3 ligands CXCL9, CXCL10, or CXCL11 (lanes 2C4). After incubation cells were relocated to a 37 water bath for 5 min. Proteins in whole cell lysates were separated by SDSCPAGE, transferred to membranes, and immunoblotted with anti-phosphotyrosine mAb. Arrows show proteins showing increased tyrosine phosphorylation after CXCR3Cligand conversation. (a) and (b) represent experiments with T cells harvested from two.