TCR-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac, and myosin IIA

Abstract

Human effector memory (EM) CD4 T cells may be recruited from the blood into a site of inflammation in response either to inflammatory chemokines displayed on or specific Ag presented by venular endothelial cells (ECs), designated as chemokine-driven or TCR-driven transendothelial migration (TEM), respectively. We have previously described differences in the morphological appearance of transmigrating T cells as well as in the molecules that mediate T cell-EC interactions distinguishing these two pathways. In this study, we report that TCR-driven TEM requires ZAP-70-dependent activation of a pathway involving Vav, Rac, and myosin IIA. Chemokine-driven TEM also uses ZAP-70, albeit in a quantitatively and spatially different manner of activation, and is independent of Vav, Rac, and mysosin IIA, depending instead on an as-yet unidentified GTP exchange factor that activates Cdc42. The differential use of small Rho family GTPases to activate the cytoskeleton is consistent with the morphological differences observed in T cells that undergo TEM in response to these distinct recruitment signals.

Figure 1

T cell ZAP-70 activity is required for both chemokine- and TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with 0, 10, 30 and 100 μg/ml piceatannol (pic) for 30 minutes or 0 and 40 μM Syk Inhibitor II for 60 minutes. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in response to chemokines on the EC; chemokine-driven TEM) at 15 min flow (pic) or 50 min flow (Syk Inhib. II) and % TEM of cells expressing Vβ2TCR (VB2+, those that interact with TSST-1 presented by the EC; TCR-driven TEM) after 50 min flow. Graphs display combined data from 2 separate experiments (pic) or one representative experiment of two (Syk inhib. II). *, p<0.001, **, p<0.0001. B. Samples from TEM experiments were stained for Vβ2TCR, NFAT, and nuclei (DAPI). Shown are samples from T cells treated with 30 and 100 μg/ml pic. Arrows point to NFAT staining in Vβ2TCR+ T cells. Note that NFAT is translocated to the nucleus in the Vβ2TCR+ T cell at 30 μg/ml pic, but is excluded from the nucleus in all Vβ2TCR− cells as well as the Vβ2TCR+ cell at 100 μg/ml pic.

Figure 2

Activated ZAP-70 in EM CD4 T cells undergoing chemokine- and TCR-driven TEM. A. EM CD4 T cells on TNF-treated CIITA HDMEC overlaid with TSST-1 after 5 minutes of flow stained for DAPI (blue), Vβ2TCR (VB2, green), and Phospho(Tyr319)ZAP-70 (P-ZAP-70, white) were imaged with a confocal microscope at different Z sections corresponding to the T cell-EC interface and above, as indicated by the numbers to the right (in μm). Z sections of the two T cells in the top panels are shown, one Vβ2TCR− (left panels) and the other Vβ2TCR+ (right panels). Note the strong symmetrical P-ZAP-70 staining pattern in the VB2+ cell at all layers, and the focal dots in the VB2− cell apparent mostly at the T cell-EC interface. B. Quantification of P-ZAP-70 staining of Vβ2TCR− (VB2−) and Vβ2TCR+ (VB2+) T cells at 5 minute flow. N=22, *, p<0.0001. C. Confocal images of a Vβ2TCR+ T cell (VB2 in red) at 30 minutes flow with a TEP that is underneath the EC monolayer (top panel), the cell body near the apical surface of the EC (middle panels) and the top of the T cell (bottom panels).

Figure 3

Phospho-LAT staining of EM CD4 T cells at 5 and 30 minutes flow. A. 5 minute samples stained with DAPI (blue), phalloidin (green), Vβ2TCR (VB2, red), and Phospho-LAT (Tyr191)(P-LAT, white). Left panels show one Z section of a Vβ2TCR− T cell at the EC interface, and the right panels show two Z sections, one at the EC interface and another 1.26 μm above. B. Quantification of P-LAT staining of Vβ2TCR− (VB2−) and Vβ2TCR+ (VB2+) T cells at 5 minute flow. N=20, *, p<0.0001. C. Confocal images of a Vβ2TCR+ T cell at 30 minutes flow with a TEP that is underneath the EC monolayer (top panels), the cell body near the apical surface of the EC (middle panels) and further above the EC interface (bottom panels). Numbers indicate μm above the TEP.

Figure 4

Activated LFA-1 and P-ZAP-70 staining of chemokine- and TCR-driven TEM at 5 minutes. Left four panels: Chemokine-driven TEM. Z sections showing DAPI, P-ZAP-70 (red), activated LFA-1 (white), and an averlay of P-ZAP-70 and activated LFA-1. Right four panels: TCR-driven TEM. Z sections showing DAPI, Vβ2TCR (green), P-ZAP-70 (red), and activated LFA-1 (white).

Figure 5

Confocal analysis of talin and RACK1 in EM CD4 T cells undergoing chemokine- and TCR-driven TEM. Left panels show Z slices of samples stained for nuclei (DAPI, blue), talin (green, Vβ2TCR (VB2, red) and activated LFA-1 (white). The right panels show Z slices of samples stained for nuclei (DAPI, blue), F-actin (phalloidin, green), Vβ2TCR (VB2, red), and RACK1 (white). The top set of panels represent Vβ2TCR− T cells at 5 min flow, the middle panels represent Vβ2TCR+ T cells at 5 min flow, and the bottom panels represent Vβ2TCR+ T cells at 30 min flow. The top panels of the 5 min flow samples are at the T cell EC interface, with the middle and bottom panels taken above, as the numbers indicate in μm. The top panels of the 30 min samples are underneath the EC monolayer, with the next three rows taken at layers above that plane, in the numbers indicated in μm.

Figure 6

Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in response to chemokines on the EC; chemokine-driven TEM) and cells with Vβ2TCR (VB2+, those that interact with TSST-1 presented by the EC; TCR-driven TEM) after 50 min flow. Graphs display data combined from 3 separate experiments. *, p<0.001. B. FACS analysis of EM CD4+ T cells transduced with recombinant fusion proteins. Filled peak denotes nontransduced cells, and lines depict cells transduced with Hph-EGFP (GFP) and Hph-EGFP-Vav dominant negative (Vav DN). C. TEM assays of EM CD4+ T cells transduced with recombinant proteins. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−)at 15 min flow and cells with Vβ2TCR (VB2+) after 50 min flow. Graphs display data combined from 3 separate experiments. *, p<0.001.

Figure 7

Chemokine-driven TEM requires Cdc42 and TCR-driven TEM requires Rac. A. FACS analysis of EM CD4 T cells transduced with recombinant fusion proteins. Filled peak denotes nontransduced cells, and lines depict cells transduced with Hph-GFP-Rac1 wild type (Rac wt), Hph-GFP-Rac dominant negative (Rac DN), Hph-GFP-Cdc42 wild type (Cdc42 wt), and Hph-GFP-Cdc42 dominant negative (Cdc42 DN). B. TEM assays of EM CD4 T cells transduced with recombinant proteins. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in response to chemokines on the EC; chemokine-driven TEM) at 15 min flow and cells with Vβ2TCR (VB2+, those that interact with TSST-1 presented by the EC; TCR-driven TEM) after 50 min flow. Graphs display data from one representative experiment of at least 3. *, p<0.001. C. TEM assays of EM CD4 T cells treated with 0, 1, and 4 μM EHop-016 20 hours, flow 50 minutes. Graphs display data combined from three experiments. *, p<0.0001.

Figure 8

Myosin IIA activity is required for TCR-driven TEM at a step after TEP formation, but not chemokine-driven TEM. A. TEM assays of EM CD4 T cells treated with blebbistatin. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in response to chemokines on the EC; chemokine-driven TEM, left graph) at 15 min flow and cells with Vβ2TCR (VB2+, those that interact with TSST-1 presented by the EC; TCR-driven TEM, middle graph) after 50 min flow, as well as %TEPs of the VB2+ cells (right graph). Graphs display data from one representative of three separate experiments. *, p<0.001. B. EM CD4 T cells treated with vehicle or blebbistatin 30 minutes on TNF-treated CIITA HDMEC overlaid with TSST-1 after 30 minutes of flow stained for DAPI (blue) and Vβ2TCR (VB2, green) were imaged with a confocal microscope at different Z sections corresponding to below the EC monolayer, first row, and the T cell body 1.5 μm above on the apical side of the EC (second row). Note the exaggerated TEP (the entire VB2 staining underneath the EC monolayer, first row) in the blebbistatin treated sample compared to vehicle. Scalebar = 20 μm. C. Quantification of TEP length in vehicle and blebbistatin treated samples at 30 min flow. N=26, *p<0.0001. D. TEM assays of EM CD4 T cells treated with vehicle or 10 μM ML-7 for 30 minutes, 50 minute flow. Graphs display data combined from three experiments. *, p=0.0005, **, p<0.0001.