Antigen presentation by human microvascular endothelial cells triggers ICAM-1-dependent transendothelial protrusion by, and fractalkine-dependent transendothelial migration of, effector memory CD4+ T cells

Abstract

TCR engagement on adherent human effector memory CD4(+) T cells by TNF-treated HUVECs under flow induces formation of a transendothelial protrusion (TEP) by the T cell but fails to induce transendothelial migration (TEM). In contrast, TCR engagement of the same T cell populations by TNF-treated human dermal microvascular cells (HDMEC) not only induces TEP formation, but triggers TEM at or near the interendothelial cell junctions via a process in which TEP formation appears to be the first step. Transduction of adhesion molecules in unactivated HDMEC and use of blocking Abs as conducted with TNF-activated HDMEC indicate that ICAM-1 plays a nonredundant role in TCR-driven TEP formation and TEM, and that TCR-driven TEM is also dependent upon fractalkine. TEP formation, dependence on ICAM-1, and dependence on fractalkine distinguish TCR-induced TEM from IP-10-induced TEM. These in vitro observations suggest that presentation of Ag by human microvascular endothelial cells to circulating CD4(+) effector memory T cells may function to initiate recall responses in peripheral tissues.

Figure 1

TCR engagement induces transendothelial protrusions in effector memory CD4+ T cells on cultured HUVEC. A. Phase contrast (PC), nuclear staining with DAPI, and immunofluorescence staining of Vβ2TCR+ (upper and middle panels) and Vβ2TCR- EM CD4+ T cell captured by a TNF-treated CIITA-transduced HUVEC monolayer preloaded with TSST-1under conditions of venular flow. Upper and lower panels taken with a 40× objective. Middle panels show deconvoluted images of a series of 1 μm Z-stack of pictures taken with a 63× objective of Vβ2TCR staining. Note that the TEP extends through the HUVEC monolayer. Lower panels show a Vβ2TCR- cell. Note the spread morphology and close proximity of the nucleus to the leading edge. B. Left graph: Flow TEM assay was performed with EM CD4+ cells for 30 and 60 minutes at 1 dyne/cm² on TNF-treated CIITA HUVECs preloaded with TSST-1, fixed, and stained for Vβ2TCR and CD3 (to detect protrusions in Vβ2TCR- cells). Mean and s.e.m. of the number of cells showing a TEP in 5 sets of 20 cells. P<0.01 for comparison of Vβ2TCR– vs Vβ2TCR+ at 60’. One representative experiment of three is shown. Right graph: Flow TEM assay was performed with EM CD4+ cells for 60 minutes at 1 dyne/cm² on TNF-treated CIITA HUVECs preloaded or not with TSST-1, fixed and stained for Vβ2TCR. Mean and s.e.m. of the number of cells showing a TEP in ten sets of 20 cells. P<0.0001. C. CD4+ cells were depleted of CD45RO+ positive cells (naïve) or depleted of CD45RA+ cells and then stained with CCR7-FITC and FACS-sorted for CCR7 high (central memory, CM) and CCR7 low (EM) and used in a one hour flow TEM assay or static TEM assay with TNF-treated CIITA HUVECs preloaded with TSST-1 and stained for Vβ2TCR. Mean and s.e.m. of number of Vβ2TCR+ cells showing a TEP in 10 sets of 20 cells. P<0.001 for comparison of EM, flow versus all other groups; P>0.05 for all other comparisons.

Figure 2

TCR-dependent TEP formation and TEM of EM CD4+ cells on cultured HDMEC. A. CIITA/TSST-1 HDMEC model. EM CD4+ T cell on TNF-treated CIITA HDMECs preloaded with TSST-1, overlaid or not with IP-10, and subjected to 15, 30, or 60 minutes flow. Left panel: mean and s.e.m. of Vβ2TCR+ cells with a TEP. No significant increase is observed between 15’ and 60’ and no effect of IP-10 is noted. Right panel: mean and s.e.m. of transmigrated cells. P<0.05 between Vβ2TCR- cells, with and without IP-10. P<0.001 between Vβ2TCR- and Vβ2TCR+ cells at 15’. P<0.001 between Vβ2TCR+ cells at 15’ compared to 60”. Note that there is a significant level of spontaneous TEM across HDMEC monolayers that is augmented by IP-10 and that TCR signals inhibit both spontaneous and IP-10-dependent TEM at 15’. However, TCR-stimulated cells do undergo TEM by 60’. B. TEP formation and TEM occurs at or near EC cell junctions. EM CD4+ cells on TNF-treated CIITA HDMECs preloaded with TSST-1 after 30 minutes flow, fixed and stained for Vβ2TCR (green), VE-cadherin (red), and DAPI (blue). Upper panels taken with a 40X objective show phase contrast and merged fluorescent staining; the top right panel is an enlarged view of the Vβ2TCR+ cell with a TEP. Lower panels show deconvoluted images (layer 8 and 20) from a series of 0.2 μm Z-stack of pictures taken with a 63X objective of Vβ2TCR staining (green) and DAPI (white) and merge of Vβ2TCR, DAPI, and VE-cadherin (red). Note that the TEP and DNA in the TEP are in focus coincident with HDMEC nuclei, but out of focus on the apical surface where the T cell body still resides. C. Shear stress augments TEP formation. EM CD4+ T cells were used in TEM assay for 15 or 30 minutes at 0 (static) or 1 dyne/cm² (flow) on TNF-treated CIITA HDMEC pre-loaded or not with TSST-1. Numbers above brackets indicate p values between groups. D. Sustained NFAT nuclear translocation in transmigrated antigen-specific EM CD4+ T cells. EM CD4+ T cells on TNF-treated CIITA HDMEC preloaded with TSST-1 were fixed and stained for Vβ2TCR (blue), NFAT1 (green), and nuclei (DAPI, red) after 60’ flow. Arrow indicates a Vβ2TCR+ cell with a TEP and NFAT in the nucleus next to a Vβ2TCR- cell with NFAT excluded from the nucleus. Other transmigrated Vβ2TCR+ cells with NFAT nuclear colocalization, in contrast to the transmigrated Vβ2TCR- cells, are also evident, for example, in the upper portion. In 10 separate fields examined, NFAT translocation to the nucleus was observed in 77/78 and 3/79 of Vβ2TCR+ and Vβ2TCR- cells, respectively.

Figure 3

Photomicroscopic analyses of TCR formation and TEM in the FcγRII HDMEC/OKT3 model. A. Quantification of TEP formation and TEM of EM CD4+ cells at the indicated times under venous flow conditions on TNF-treated, FcγRII HDMEC preloaded with OKT3 mAb. Samples were fixed and stained with anti-CD3-FITC and DAPI and analyzed as described in Materials and Methods. Mean and s.e.m. of % of cells with a TEP and % TEM (left and right graph, respectively) from 10 groups of 20 cells each. P<0.001 for comparison of all TEM groups. B. Time lapse fluorescence and fluorescence deconvolution photomicroscopic analysis of TEP formation. PKH26-labeled EM CD4+ T cells on FcγRII HDMEC preloaded with OKT3 mAb, under flow. Pictures were taken once every minute using a 10× objective and TRITC filter (upper panels). Shown are pictures corresponding to 6−45 minutes presented from left to right in each row. At the end of the flow assay, the sample was mounted on a slide in DAPI-containing mounting medium, and a series of Z-stack images were taken using a 63× objective and a DAPI filter at 0.3 μm intervals and deconvoluted. Lower panel shows two slices, 13 slices apart, from the deconvoluted images. The arrow points to the cell that had formed a TEP in the upper panels. Note that the EC nuclei and the nucleus of the T cell exhibiting a TEP are in the same plane of focus, whereas the nucleus of the other T cell in the same field that had not formed a TEP remains in a different plane, corresponding to the apical surface of the EC, as determined by phase contrast optics (not shown).

Figure 4

TEM and TEP formation in flow assays using TSST-1 preloaded CIITA HDMECs co- transduced with E-selectin, ICAM-1, VCAM-1, or all three (EIV). A. FACS analysis of transductants. B. % of Vβ2TCR+ cells (Vβ2+, IP10, 15 and 60’) with a TEP. C. % TEM of EM CD4+ cells used in 15’ and 60’ flow assay with CIITA co-transductants preloaded with TSST-1 and overlaid or not with IP-10, fixed and stained for Vβ2TCR and nuclei (DAPI). Left graph shows Vβ2TCR- cells with or without IP-10 at 15 minutes. Right graph shows Vβ2TCR+ cells (with IP-10) at 15 and 60 minutes. Mean and s.e.m. from data combined from two separate experiments are shown. P<0.001 between 15 and 60 minutes for Vβ2TCR+ cells on EIV, ICAM-1 and VCAM-1 transductants.

Figure 5

Analysis of signals that contribute to antigen-driven TEM. A. Antigen-driven TEM is pertussis toxin sensitive. EM CD4+ T cells were treated with vehicle, LY294002 (LY), or pertussis toxin (ptx) prior to flow assays on TNF-treated CIITA HDMEC preloaded with TSST-1. Shown are the mean and s.e.m. of % TEM (left graph; P<0.001 for comparison of Vβ2TCR- cells ptx, 15’ compared to vehicle and LY 15’; P<0.001 for comparison of Vβ2TCR+ cells ptx, 60’ compared to vehicle and LY 60’) and % TEP of Vβ2TCR+ cells (right graph; P<0.001 for comparison of vehicle to ptx at 15’; P<0.05 for comparison of vehicle to ptx at 60’; P>0.05 for comparisons of vehicle vs LY at both 15’ and 60’).

Figure 6

Both anti-ICAM-1 and anti-fractalkine antibodies block TCR-driven TEM, but only anti-ICAM-1 antibody blocks TEP formation. EM CD4+ T cells used in TEM assay on TNF-treated CIITA HDMEC preloaded with TSST-1 and incubated with IgG, anti-E-selectin, anti-ICAM-1, anti-VCAM-1 or anti-fractalkine blocking antibody. Shown are the mean and s.e.m. of % TEM (left graph; P<0.001 (**) between Vβ2TCR+ cells at 60’ on HDMEC treated with IgG or with anti-ICAM-1 or anti-fraktalkine Ab) and % TEP of Vβ2TCR+ cells (right graph; P<0.05 (*) between cells at 60’ on HDMEC treated or not with anti-ICAM-1 or anti-fractalkine Ab).