LFA-1 Ligation by High-Density ICAM-1 Is Sufficient To Activate IFN-γ Release by Innate T Lymphocytes

Akshat Sharma, Stephanie M. Lawry, Bruce S. Klein, Xiaohua Wang, Nathan M. Sherer, Nicholas A. Zumwalde and Jenny E. Gumperz
J Immunol October 15, 2018, 201 (8) 2452-2461; DOI: https://doi.org/10.4049/jimmunol.1800537

Abstract

By binding to its ligand ICAM-1, LFA-1 is known to mediate both adhesion and costimulatory signaling for T cell activation. The constitutively high LFA-1 cell surface expression of invariant NKT (iNKT) cells has been shown to be responsible for their distinctive tissue homing and residency within ICAM-rich endothelial vessels. However, the functional impact of LFA-1 on the activation of iNKT cells and other innate T lymphocyte subsets has remained largely unexplored. In particular, it is not clear whether LFA-1 contributes to innate-like pathways of T cell activation, such as IFN-γ secretion in response to IL-12. Using a recombinant ICAM-1-Fc fusion protein to stimulate human iNKT cells in the absence of APCs, we show that LFA-1 engagement enhances their IL-12–driven IFN-γ production. Surprisingly, exposure to high densities of ICAM-1 was also sufficient to activate iNKT cell cytokine secretion independently of IL-12 and associated JAK/STAT signaling. LFA-1 engagement induced elevated cytoplasmic Ca2+ and rapid ERK phosphorylation in iNKT cells, and the resulting IFN-γ secretion was dependent on both of these pathways. Analysis of freshly isolated human PBMC samples revealed that a fraction of lymphocytes that showed elevated LFA-1 cell surface expression produced IFN-γ in response to plate-bound ICAM-1–Fc. A majority of the responding cells were T cells, with the remainder NK cells. The responding T cells included iNKT cells, MAIT cells, and Vδ2+ γδ T cells. These results delineate a novel integrin-mediated pathway of IFN-γ secretion that is a shared feature of innate lymphocytes.

Introduction

T cells are typically considered to epitomize adaptive immunity. However, it has recently become clear that a fraction of T lymphocytes shares with innate lymphocytes the expression of a master transcription factor, promyelocytic leukemia zinc finger (PLZF) (1). PLZF is required for the proper development of innate lymphoid cells and human NK cells and is expressed in both of these subsets in the periphery (2, 3). Thus, PLZF is associated with an innate functional status of lymphocytes. The best known PLZF+ T cells are invariant NKT (iNKT) cells (4–6). iNKT cells use a canonical TCRα-chain rearrangement that is paired with a limited set of TCRβ-chains, recognize conserved lipid Ags presented by nonclassical CD1d Ag-presenting molecules, and have innate-like functional properties, including mediating rapid effector cytokine responses upon primary challenge (7–10). Additional subsets of T lymphocytes now known to express PLZF include mucosal-associated invariant T (MAIT) cells and certain γδ T cells (11, 12). These subsets resemble iNKT cells in that they use canonical TCR rearrangements, recognize conserved nonclassical Ags, and have innate-like functional properties (13–18). Thus, based on their constrained TCR structures, specificity for conserved ligands, and shared transcriptional program, these T cell subsets can be grouped into a distinct compartment called innate T lymphocytes (1, 19, 20). The specific features of innate T cells that are conferred by their shared expression of PLZF, and that may thus set them apart as a group from adaptive T lymphocytes, remain largely unexplored.

One such distinctive characteristic conferred by PLZF is upregulated expression of the integrin LFA-1 (21). LFA-1 plays critical roles in T cell migration via binding to its adhesion ligand ICAM-1, which is expressed on vascular endothelium and other cell types (22) The elevated LFA-1 expression of murine iNKT cells has been shown to be responsible for their stable residency in the sinusoids of the liver, which are endothelial vessels that are high in ICAM-1 (21, 23). Similarly, under steady-state conditions, iNKT cells have been observed by intravital microscopy to constitutively patrol other ICAM-rich areas of the vasculature, including pulmonary endothelial surfaces (24). Additionally, both human and murine iNKT cells have been found to be recruited to atherosclerotic plaques, which are inflamed vascular endothelial areas where ICAM-1 levels may be elevated (25–29). Thus, the elevated LFA-1 expression level of iNKT cells likely plays a key role in their distinctive tissue recruitment and residency patterns. However, what has been less clear is whether their high LFA-1 status impacts the functional responses of iNKT cells.

LFA-1 plays a key role during TCR-mediated activation. TCR signaling from initial Ag recognition events induces the unfolding of LFA-1 from its low-affinity state into higher affinity conformations that are able to bind to ICAM-1 (30). LFA-1 binding to ICAM-1 leads to the rapid activation of Src family kinases (e.g., Lck, ZAP-70) and an ensuing signaling cascade resulting in the activation of PLCγ and triggering of downstream Ca2+ signaling (31–33). Ultimately, the signaling events contributed by LFA-1 are thought to lower the threshold of Ag required for productive T cell activation (34, 35). iNKT cells are known to recognize certain unusual glycolipids, such as α-galactosylceramide (α-GalCer), as highly potent TCR agonists (9, 36, 37). However, it is not clear that many pathogenic microbes produce α-GalCer or closely related lipids, and lipids of this type derived from other physiological sources appear to be present in only extremely small quantities (38, 39). In contrast, other highly abundant lipid species (e.g., lyso-phosphatidylcholine and β-linked glycosylsphingolipids) serve as weak TCR agonists for iNKT cells (40–43). Thus, because iNKT cells probably experience only low levels of TCR stimulation in most physiological settings due to a scarcity of high-affinity and/or an abundance of low-affinity Ags, LFA-1–mediated costimulation likely plays an important role during their TCR-mediated activation.

Less clear is whether LFA-1 also impacts other pathways of iNKT cell activation. For example, iNKT cells can become activated directly by proinflammatory cytokines (e.g., IL-12) without requiring a concurrent TCR signal (44–47). Indeed, prior studies in murine models of bacterial infection have suggested that TCR signaling is not required (48), and instead the cytokine-dependent pathway of activation is a dominant means by which iNKT cells become activated (49–51). It has been established that the IFN-γ produced by adaptive T cells in response to TCR stimulation is secreted in a directional manner toward the immunological synapse, which is also where LFA-1 is aggregated at the cell surface (52), and we have observed a similar phenomenon for iNKT cells responding to DCs presenting the potent lipid Ag α-GalCer (Supplemental Fig. 1A). However, in the case of TCR-independent IFN-γ secretion by iNKT cells, it is not clear whether cell contact, and thus LFA-1 engagement, plays any role. This is an important question because the physiological effects of IFN-γ secretion probably depend largely on the identity and functional characteristics of the recipient cells, and thus IFN-γ that is produced in the context of an immunological synapse with an APC may serve a different purpose than IFN-γ that is produced during nonsynaptic contact with other cells or that is produced in a manner that is independent of cell contact.

We previously observed that cytokine-stimulated human iNKT cells secreted substantially more IFN-γ when B lymphoblastoid cells were also present, even if the B lymphoblastoid cells lacked CD1d and thus were not able to deliver TCR stimulation to the iNKT cells (47). Moreover, the IFN-γ produced by iNKT cells during exposure to IL-12 on an ICAM-1–coated surface appeared to colocalize intracellularly with the tubulin nexus representing the microtubule organizing center (Supplemental Fig. 1B), which has been shown in migrating T cells to be positioned adjacent to the area of the plasma membrane where LFA-1 is aggregated (53). Finally, it has been observed through intravital microscopic analysis that i.v. injection of the cytokines IL-12 and IL-18 leads to rapid IFN-γ production by the iNKT cells patrolling hepatic sinusoids (54). Intriguingly, the iNKT cell response in this situation was accompanied by migration arrest, suggesting that it was associated with conformational changes and signaling by LFA-1. Together, these observations suggested to us that LFA–ICAM interactions might facilitate or enhance TCR-independent IFN-γ secretion by iNKT cells. Therefore, in this analysis, we set out to investigate the impact of LFA-1 engagement on TCR-independent IFN-γ secretion by iNKT cells.

Materials and Methods

PBMC isolation

Venous blood was obtained from healthy adult male or female subjects in accordance with a University of Wisconsin Institutional Review Board–approved protocol. Freshly drawn blood samples were mixed with heparin sodium (10 U/ml of blood) (Sagent Pharmaceuticals), and mononuclear cells were purified by density gradient centrifugation (Ficoll-Paque Premium; GE Healthcare).

iNKT cells

Human iNKT cells used in these analyses included clonal lines that we have previously established (40, 49, 55–57) and polyclonal cultures resulting from short-term in vitro expansion of cells sorted using α-GalCer–loaded CD1d tetramer provided by the NIH Tetramer Core Facility at Emory University, as we have previously described (58, 59). Clonal and polyclonal iNKT cell cultures were maintained in culture medium comprised of RPMI 1640 diluted with glucose-free RPMI (Biological Industries) to yield a final glucose concentration of 7.5 mM, 3% human AB serum (Atlanta Biologicals), 10% heat-inactivated bovine calf serum (HyClone), 1% penicillin-streptomycin, 1% l-glutamine, and 200 U/ml IL-2 (PeproTech). iNKT cultures used for experiments were of 98–100% purity as assessed by flow cytometric analysis with α-GalCer–loaded CD1d tetramers.

ICAM-1 stimulation of iNKT cells

High protein–binding 96-well plates (Corning) were coated with recombinant human ICAM-1–Fc (Acro Biosystems) or an isotype-matched negative control mAb (clone MOPC21; Sigma-Aldrich), then washed with PBS to remove unbound ICAM-1. iNKT cells (50 × 105 per well) were added in culture medium lacking IL-2. Where indicated, the following compounds were included in the culture medium: 20 U/ml recombinant human IL-12p70 (PeproTech); the indicated concentrations of U0126 (InvivoGen) or cyclosporin A (Sigma-Aldrich); 1 μg/ml anti-human CD11a blocking mAb (clone HI111; Invitrogen); 1 μg/ml anti-human CD18 blocking mAb (Clone TS1/18; BioLegend); 5 μg/ml anti-CD1d blocking mAb (clone CD1d55); or the indicated concentrations of JAK inhibitor I (Calbiochem). Where indicated, instead of ICAM-Fc simulation, the iNKT cells were exposed to plate-bound CD1d-Fc that had been pulsed with 25 ng/ml α-GalCer as previously described (40). Each stimulation condition was performed in three to four replicate wells. iNKT cells were stimulated for 18–24 h at 37°C and 5% CO2, then culture supernatants were harvested and assayed for IFN-γ using a sandwich ELISA (capture Ab clone MD-1 from BioLegend; biotinylated detection Ab clone 4SB.3 from BD Pharmingen). IFN-γ concentrations were determined by comparison with a standard curve of recombinant human IFN-γ (PeproTech) assayed in parallel.

Microscopic analysis of iNKT cell cytoplasmic Ca2+

iNKT cells were labeled with Fluo-4 (Calbiochem) according to the manufacturer’s instructions, then 7.5 × 104 cells were seeded onto glass chamber slides (ibidi) coated with a 1 μg/ml or a 5 μg/ml solution of ICAM-1–Fc and blocked with 2.5% BSA or coated with poly-l-lysine. The slides were placed into a 37°C and 5% CO2 chamber, and images were taken every 20 s for 30 min using a Nikon Ti-Eclipse inverted wide-field microscope. For analysis of Fluo-4 signal intensity, 25 iNKT cells from each condition were chosen randomly at a single time point based on bright field images, and the fluorescence intensity of each cell was determined using FIJI ImageJ2 software (imagej.net/Fiji). For analysis of Fluo-4 signal over time, live cells were manually tracked over 30 min (90 frames) with the changes in two-dimensional mean fluorescence intensity (MFI) gauged on a per cell basis.

Detection of ERK phosphorylation

Jurkat T cells (2 × 106) or iNKT cells (1.5 × 106) were stimulated with anti-human CD11a mAb (Clone TS2/4; BioLegend) for the indicated times in culture medium lacking IL-2. Cells were lysed using CelLytic-M (Sigma-Aldrich) buffer infused with one tablet of PhosSTOP (Roche) phosphatase inhibitor. Lysates were run on a 12% SDS-PAGE gel, then transferred onto a polyvinylidene difluoride membrane using a Trans-Blot Turbo semidry transfer system (Bio-Rad). Membranes were blocked overnight at 4°C in a solution of TBS, 0.05% Tween-20, 5% nonfat dry milk (Sanalac), and 5% BSA (Fisher Scientific). Phosphorylated or total ERK bands were detected using a polyclonal rabbit anti-human pERK Ab (catalog no. 9101S; Cell Signaling Technology) or polyclonal rabbit anti-human MAPK p44/42 ERK1/2 Ab (catalog no. 9102S; Cell Signaling Technology), followed by detection using a goat anti-rabbit HRP-linked secondary Ab (catalog no. 7074S; Cell Signaling Technology). Membranes were developed with Clarity Western ECL Substrate (Bio-Rad) and analyzed using a VersaDoc imaging system (Bio-Rad). Total and pERK band intensities were quantitated using FIJI ImageJ software. For flow cytometric detection of ERK phosphorylation, iNKT cells (1 × 106) were stimulated with anti-human CD11a mAb (clone: TS2/4; BioLegend) for 5 min, followed by fixation using BioLegend’s Fixation Buffer (catalog no. 420801) and permeabilized with chilled True-Phos buffer (BioLegend). Cells were then stained with anti-pERK mAb (clone: 4B11B69; BioLegend) or isotype control mAb for 30 min in darkness at room temperature. Samples were read on an LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).

ICAM-Fc stimulation of fresh PBMCs

PBMCs (1 × 106) were incubated for 18–24 h with plate-bound ICAM-1–Fc (coated at 5 μg/ml) or with an isotype-matched negative control mAb (5 μg/ml MOPC-21; Sigma-Aldrich). PBMCs were resuspended, and Fc receptors were blocked by incubation in a solution of PBS containing 25% human AB serum (Atlanta Biologicals) for 20 min at 4°C, then cell surface staining was performed for 30 min at 4°C using the following human-specific Abs or tetramers: anti-CD3 (OKT3; BioLegend), anti-CD4 (OKT4; BioLegend), anti-CD8α (HIT8a; BioLegend), anti-CD8β (SIDI8BEE; eBioscience), anti-CD11a (HI111; BioLegend), anti-CD56 (5.1H11; BioLegend), CD1d tetramer (PBS-57–loaded; NIH Tetramer Core Facility), MR1 tetramer (5-OP-RU–loaded; NIH Tetramer Core Facility), and anti-Vδ2 TCR (123R3; Miltenyi Biotec). Cells were fixed and permeabilized according to the manufacturer’s instructions using the BD Cytofix/Cytoperm kit (BD Biosciences), then stained with anti–IFN-γ (4S.B3; BioLegend), anti-PLZF (Mags.21F7; eBioscience), or the respective negative control mAbs suggested by the vendor. Cells were then washed, resuspended in PBS, and analyzed on an LSR II flow cytometer (BD Biosciences). Staining data were analyzed using FlowJo analysis software (Tree Star).

Statistical analyses

An unpaired two-sided Student t test was used to assess replicates from different experimental conditions. A Mann–Whitney U test was used to assess sample groups comprised of data aggregated from multiple independent analyses. Sets of paired samples were analyzed using a Wilcoxon matched pairs analysis.

Results

ICAM-1 binding to LFA-1 costimulates cytokine-driven IFN-γ secretion by human iNKT cells

Because it was previously shown in a murine model system that PLZF confers elevated LFA-1 expression, and murine iNKT cells have constitutively high levels of this integrin (21), we first sought to confirm whether human iNKT cells also show high expression of LFA-1. PBMCs purified from healthy adult subjects were stained for CD3 to identify T cells and costained for CD11a to detect LFA-1 and for CD1d tetramer to distinguish iNKT cells. Flow cytometric analysis revealed that the iNKT cells were among the brightest CD11a-expressing T cells (Fig. 1A, left plots). Analysis of samples from multiple unrelated donors revealed that this was consistently the case, with the iNKT cell subset showing on average ∼1.7-fold brighter CD11a staining than tetramer-negative T cells (Fig. 1A, right plot).

FIGURE 1.
FIGURE 1.

Elevated LFA-1 expression on human iNKT cells costimulates IFN-γ secretion in response to IL-12p70. (A) Freshly isolated human PBMCs were stained with Abs against CD3 and CD11a and with α-GalCer–loaded CD1d tetramer and analyzed by flow cytometry. Plots on left show CD11a expression by CD1d tetramer–positive (black line) compared with tetramer–negative (gray line) for one representative experiment. Plot on right shows results from analysis of PBMC samples from five unrelated healthy adults. MFI of CD11a staining is plotted for CD1d tetramer positive (iNKT cells) and tetramer-negative (non-iNKT cells). (B) iNKT cells were incubated for 24 h in medium containing 20 U/ml recombinant human IL-12p70, in the presence of plate-bound ICAM-1–Fc (coated at 5 μg/ml) or negative control mAb (IL-12 alone). Secreted IFN-γ was quantitated using a standardized ELISA. The plot shows aggregated results from seven independent experiments using five different iNKT cell clonal lines (clones PP1.2, PP1.3, PP1.10, J3N.5, GG1.2). (C) iNKT cells were incubated for 24 h in the following conditions: 1) culture medium alone (No stim), 2) medium containing IL-12p70 in wells coated with a negative control mAb (IL-12 alone), or 3) medium containing IL-12p70 in wells coated with ICAM-1–Fc. The incubation in the third condition was performed in the presence or absence of the indicated blocking Abs. Secreted IFN-γ was quantitated by ELISA. The plot shows results from one representative experiment out of two using a short-term in vitro expansion of polyclonal iNKT cells (318D line); bars indicate means and SD from four replicates per treatment. Similar results were observed in two additional independent experiments (once with the 318D line, and once with iNKT clone PP1.2).

We next investigated the impact of ICAM-1 exposure on iNKT cell IFN-γ secretion in response to IL-12. Human iNKT cells were incubated in culture medium containing recombinant human IL-12p70 in the presence or absence of plate-bound ICAM-1–Fc for 24 h. Culture supernatants were harvested and tested for secreted IFN-γ by ELISA. Although exposure to IL-12 alone was sufficient to induce iNKT cell IFN-γ secretion, the IFN-γ amounts were consistently elevated in the presence of plate-bound ICAM-1–Fc (Fig. 1B), suggesting that ICAM-1 exposure costimulated the IL-12–driven IFN-γ production. Because LFA-1 is comprised of two subunits, CD11a (integrin α L) and CD18 (integrin β 2), we tested the involvement of each of the subunits. iNKT cells were incubated with IL-12 and plate-bound ICAM-1–Fc in the presence or absence of blocking Abs against CD11a and CD18. Blockade of either CD11a or CD18 prevented the ICAM-dependent enhancement of iNKT cell IFN-γ secretion, and anti-CD11a blockade completely abrogated the response (Fig. 1C). Together, these results demonstrate that ICAM-1 binding to LFA-1 costimulates iNKT cell IFN-γ secretion that is driven by IL-12.

LFA-1 stimulation alone is sufficient to induce iNKT cell IFN-γ secretion

To further investigate the requirements for ICAM-1 to promote IFN-γ secretion by iNKT cells, we titrated the density of the plate-bound ICAM-1–Fc. Low densities of ICAM-1–Fc showed no detectable costimulatory effect, but as the density increased, we observed an abrupt enhancement of iNKT cell IFN-γ secretion in response to IL-12 (Fig. 2A, gray circles). Surprisingly, however, at densities of ICAM-1–Fc above this inflection point, we observed that iNKT cell IFN-γ secretion was also induced even in the absence of IL-12 (Fig. 2A, black triangles). Addition of even high doses of an inhibitor of JAK, which is required for IL-12–mediated activation of iNKT cells, did not abolish the iNKT cell IFN-γ secretion induced by exposure to high-density ICAM-1–Fc, whereas it did abrogate the iNKT cell response to IL-12 cytokine (Fig. 2B). These observations suggested that ICAM-1 exposure drives IFN-γ secretion by iNKT cells directly via signaling through LFA-1 in a manner that does not require signaling through a JAK2-dependent pathway.

FIGURE 2.
FIGURE 2.

Exposure to a high density of ICAM-1 promotes iNKT cell IFN-γ secretion in a manner that is independent of IL-12 stimulation and CD1d recognition. (A) iNKT cells were incubated for 24 h in wells coated with the indicated titrated doses of ICAM-1–Fc in the presence or absence of 20 U/ml IL-12p70. Secreted IFN-γ was quantitated by ELISA. The plot shows results from one representative experiment out of two using a short-term in vitro expansion of polyclonal iNKT cells (FoB line); symbols indicate means and SD, four replicates per treatment. Similar results were observed in an additional independent experiment using iNKT clone PP1.2. (B) Left plot, iNKT cells (318D short-term polyclonal line) were incubated in wells coated with ICAM-1–Fc in the presence of the indicated concentrations of JAK2 inhibitor, and secreted IFN-γ was quantitated by ELISA; similar results were obtained using a different polyclonal iNKT line (4LF). Right plot, iNKT cells (318D short-term polyclonal line) were exposed to plate-bound anti-CD3 mAb or cultured in medium containing IL-12p70 in the presence or absence of 10 μM JAK2 inhibitor. (C) iNKT cells (318D short-term polyclonal line) were incubated in wells coated with CD1d-Fc fusion protein that had been pulsed with 25 ng/ml α-GalCer or vehicle or in wells coated with 5 μg/ml ICAM-1–Fc. Where indicated, the CD1d- or ICAM-mediated iNKT cell stimulation was performed in the presence of an anti-CD1d blocking mAb.

To confirm that CD1d recognition is not required for iNKT cell activation by ICAM-Fc, we tested the effect of inclusion of an anti-CD1d blocking Ab. Whereas the presence of an anti-CD1d mAb significantly reduced iNKT cell IFN-γ secretion in response to plate-bound CD1d-Fc pulsed with the α-GalCer lipid Ag, there was no detectable impact on iNKT cell activation by plate-bound ICAM-Fc (Fig. 2C). We also noted that most of the cultured iNKT cells used for these experiments showed no detectable cell surface expression of CD1d (Supplemental Fig. 2A), and we have not observed α-GalCer lipid Ag-mediated activation of cultured iNKT cells in the absence of CD1d+ APCs (Supplemental Fig. 2B). Thus, exposure to high-density ICAM-1 appears to activate iNKT cells independently of CD1d recognition.

LFA-1 ligation has been shown to induce reorganization of the actin cytoskeleton in T cells that leads to both Ca2+ signaling and JNK activation (33, 35). Therefore, we assessed cytoplasmic calcium levels in iNKT cells exposed to an ICAM-1–coated surface. iNKT cells were cytoplasmically labeled with Fluo-4, a dye that increases its fluorescence in proportion to the surrounding Ca2+ concentration, and placed onto glass slides coated with ICAM-1–Fc and blocked with poly-l-lysine. Microscopic imaging revealed that iNKT cells placed on slides coated either with poly-l-lysine alone or with a negative control Ig showed little evidence of Ca2+ signaling (Fig. 3A, 3B). Exposure to slides coated with 1 μg/ml ICAM-1–Fc resulted in only a minimal increase in Fluo-4 signal, but iNKT cells placed on slides coated with 5 μg/ml ICAM-1–Fc showed significantly elevated Fluo-4 signal (Fig. 3A, 3B). Moreover, analysis of the Fluo-4 signal intensity over time for a selection of individual iNKT cells revealed greater spiking of Fluo-4 signal in the high-density ICAM-1–exposed iNKT cells compared with those in the poly-l-lysine control condition (Fig. 3C). To assess the importance of Ca2+ signaling for IFN-γ production by iNKT cells in response to high-density ICAM-1, we tested the effect of cyclosporine A (CsA), a drug that not only inhibits the Ca2+-dependent serine phosphatase calcineurin but also blocks the activation of JNK and p38 MAPK signaling pathways (60). iNKT cells were exposed to plate-bound ICAM-1–Fc or anti-CD3 mAb as a positive control in the presence of titrated concentrations of CsA. IFN-γ secretion in response to either anti-CD3 or ICAM-1 was completely abrogated in the presence of CsA (Fig. 3D). Together, these results suggest that LFA-1 engagement by a high density of ICAM-1 induces signaling in iNKT cells that leads to IFN-γ production.

FIGURE 3.
FIGURE 3.

Exposure to high density ICAM-1 induces Ca2+ signaling in iNKT cells. (A–C) Cultured human iNKT cells (318D short-term polyclonal line) labeled with the calcium indicator dye Fluo-4 were placed on slides coated with the concentrations of ICAM-1–Fc or with 5 μg/ml negative control Ig or with poly-l-lysine alone. Images were taken at 20 s intervals using a fluorescence microscope. (A) A fluorescence microscopic image at original magnification ×20 for each of the conditions. (B) Results from quantitation of the fluorescence signal for 24 cells per condition. Cells were randomly chosen for Fluo-4 signal quantitation based on bright field images. (C) The Fluo-4 signal intensities over time for individual iNKT cells (indicated by different color lines) placed on slides coated with 5 μg/ml ICAM or poly-l-lysine alone. (D) iNKT cells were incubated for 24 h with plate-bound ICAM-1–Fc (coated at 5 μg/ml) or with 0.5 μg/ml soluble anti-CD3 mAb in the presence of the indicated concentrations of CsA, and secreted IFN-γ was quantitated by ELISA. The plot shows the amount of IFN-γ as a percentage of the amount produced in each condition in the absence of CsA; symbols represent the means and SD (not always visible on the scale shown) of four replicates per condition. Similar results were obtained in four independent analyses.

We previously observed that contact with APCs potentiates iNKT cell secretion of IFN-γ in response to subsequent IL-12 exposure, and this effect is highly dependent on activation of ERK in the iNKT cells (46). To investigate whether ERK signaling may play a role in LFA-1–mediated activation of iNKT cells, we first confirmed that anti-CD11a mAb stimulation is sufficient to induce ERK phosphorylation in Jurkat T cells. Jurkat cells were exposed to anti-CD11a mAb stimulation for varying amounts of time, then Western blotting of lysates was performed to detect total ERK or pERK. As shown in Fig. 4A, anti-CD11a mAb treatment resulted in ERK phosphorylation, with signals peaking after 6–9 min of stimulation and then declining. We also observed pERK after stimulation of primary PBMCs directly ex vivo, and the pERK signal was selectively inhibited in the presence of U0126 (Fig. 4B). Stimulation of cultured human iNKT cells with an anti-CD11a mAb reproducibly resulted in ERK phosphorylation as detected by Western blotting, and this was abrogated by U0126 treatment (Fig. 4C). Moreover, intracellular flow cytometric analysis confirmed that CD11a-stimulated iNKT cells showed specific staining for pERK (Fig. 4D). To evaluate whether ERK phosphorylation was required for iNKT cell IFN-γ secretion in response to ICAM-1, iNKT cells were incubated on ICAM-1–Fc–coated plates or with anti-CD3 mAb in the presence of titrated concentrations of U0126. Whereas the iNKT cell IFN-γ response to anti-CD3 mAb stimulation was partially inhibited by U0126, the response to plate-bound ICAM-1–Fc was almost completely abrogated by the presence of U0126 (Fig. 4E). Thus, compared with TCR activation, iNKT cell IFN-γ secretion in response to LFA-1 stimulation appears to be more highly dependent on ERK signaling.

FIGURE 4.
FIGURE 4.

iNKT cell IFN-γ secretion in response to LFA-1 stimulation is dependent on ERK phosphorylation. (A) Jurkat T cells were stimulated with anti-CD11a mAb for the indicated times, then lysed and subjected to Western blotting using detection Abs against either total ERK or pERK. Images at the top show the pERK and corresponding total ERK results from a representative Western blot analysis; the plot below shows the means and SD of the pERK signal normalized by the corresponding total ERK signal from three replicate Western blot analyses. (B) Freshly isolated PBMCs were stimulated for 5 min with anti-CD11a in the presence or absence of 5 μM of the MEK inhibitor U0126. Images at the top show pERK and corresponding total ERK Western blot results. The plot below shows the normalized signal of the pERK band divided by that of the corresponding total ERK band (symbols represent mean ± SD for three replicate lanes). (C) Cultured iNKT cells (FoB short-term polyclonal line) were stimulated with anti-CD11a mAb for 5 min in the presence or absence of 5 μM U0126 and lysates were Western blotted to detect pERK. Positive control indicates Jurkat cells that were stimulated with 20 nM 12-O-tetradecanoyl-phorbol-13-acetate for 10 min. Plot below shows the aggregated results from four independent analyses, with band intensities for iNKT cell pERK normalized by the signal from the corresponding positive control band run in parallel. (D) iNKT cells (318D short-term polyclonal line) were stimulated with anti-CD11a mAb for 5 min or mock-treated, then fixed and permeabilized, stained using an Ab against pERK or an isotype-matched negative control, and analyzed by flow cytometry. (E) iNKT cells (FoB short-term polyclonal line) were incubated for 24 h with plate-bound ICAM-1–Fc or with soluble anti-CD3 mAb in the presence of the indicated concentrations of U0126, and secreted IFN-γ was quantitated by ELISA. The plot shows the amount of IFN-γ as a percentage of the amount produced in each condition in the absence of U0126; symbols represent the means and SD (not always visible on the scale shown) of four replicates. Similar results were obtained in two additional independent analyses using the FoB polyclonal and PP1.3 clonal iNKT cell lines.

ICAM-1 exposure induces IFN-γ production by human innate lymphocytes directly ex vivo

We next investigated whether exposure to an ICAM-coated surface was sufficient to induce IFN-γ production by primary cells. Freshly isolated human PBMCs were incubated for 14–24 h (the final 12 h in the presence of monensin) in culture wells coated with 5 μg/ml ICAM-1–Fc or with an isotype-matched negative control mAb. The cells were resuspended, stained for cell surface markers, then fixed and permeabilized and stained to assess intracellular IFN-γ. Flow cytometric analysis revealed that a small fraction (typically 0.5–1%) of the lymphocyte population produced IFN-γ after exposure to plate-bound ICAM-1–Fc but not to the isotype-matched control mAb (Fig. 5A). The cells that produced IFN-γ after ICAM exposure had higher expression of CD11a (LFA-1) compared with those that did not respond (Fig. 5B); although, notably, not all of the CD11ahi cells showed IFN-γ production (Fig. 5A). Similar to what we had observed for cultured iNKT cells, this response was abrogated by the presence of either CsA or U0126 (Fig. 5C, 5D).

FIGURE 5.
FIGURE 5.

Exposure to high density ICAM-1 activates IFN-γ production by a small subset of human lymphocytes directly ex vivo. (A) Freshly isolated human PBMCs were incubated in culture wells coated with 5 μg/ml ICAM-1–Fc or with an isotype-matched negative control mAb, then subjected to flow cytometric analysis to assess CD11a expression levels and intracellular IFN-γ. Plots show results from analysis of the lymphocyte gate as assessed by forward and side scatter. (B) Aggregated results from analyses of PBMCs from six unrelated healthy adults, showing CD11a geometric MFI (gMFI) for the lymphocyte population that stained positively for IFN-γ compared with those that did not. (C and D) Freshly isolated human PBMCs were incubated in culture wells coated with 5 μg/ml ICAM-1–Fc in the presence of 5 μM CsA (C) or 5μM U0126 (D) or vehicle alone and then subjected to flow cytometric analysis to assess CD11a expression levels and intracellular IFN-γ.

We used t-distributed stochastic neighbor embedding (tSNE) dimensionality reduction of a series of multiparameter flow cytometric analyses to assess the PBL populations containing the responding cells. Because we had observed that the cells that produced IFN-γ in response to plate-bound ICAM-1–Fc had elevated CD11a expression, we mapped the levels of CD11a staining of cell types within the PBL gate. Whereas B cells showed CD11a staining that was on the low end of the spectrum, and most CD4 T cells expressed moderate CD11a levels, high levels of CD11a were predominant on NK cells, CD8αα T cells, a fraction of CD8β T cells, and double-negative T cells (Fig. 6A, left plots). Analysis of the specific innate lymphocyte populations (NK cells, iNKT cells, MAIT cells, Vδ2+ T cells) within PBMC samples of a group of unrelated healthy adults revealed that each of these subsets had significantly elevated CD11a expression compared with the total T cell population (Fig. 6A, right plot). Thus, for our further analyses, we gated on CD11ahi lymphocytes.

FIGURE 6.
FIGURE 6.

The ICAM-1-responsive cells include NK cells and innate T cells. Freshly isolated human PBMCs were incubated in wells coated with 5 μg/ml ICAM-1–Fc, then analyzed by flow cytometry to characterize the IFN-γ–producing cells. (A) Left plots, A freshly isolated PBMC sample was stained with Abs against CD3, CD4, CD8α, CD8β, CD56, and CD11a and analyzed by tSNE dimensionality reduction. Plot on left shows how lymphocyte subsets within the sample were clustered by the tSNE analysis. Neighboring plot shows the relative expression levels of CD11a for the cells included in the same tSNE analysis. Graph on far right shows the aggregated results from analysis of CD11a expression levels on the indicated lymphocyte subsets from PBMC samples of unrelated healthy adult donors. Each symbol represents the gMFI of the CD11a staining for the indicated cell type normalized by the CD11a gMFI of the total T cell population from the same PBMC sample. (B) Freshly isolated PBMC samples were exposed ex vivo to plate-bound ICAM-1–Fc, then stained with Abs against CD3, CD8β, CD56, CD161, and CD11a, and fixed and permeabilized and stained for intracellular IFN-γ and PLZF. Flow cytometric staining results from three independent PBMC samples were concatenated, and the CD11ahi lymphocytes were subjected to tSNE analysis. Far left plot shows the distribution of NK cells, CD56+CD161+ T cells, and IFN-γ+ cells (light gray in background corresponds to CD8β+ T cells; dark gray corresponds to all other cells). Neighboring plot shows the distribution of PLZF+ and IFN-γ+ cells. Scatter plot on right shows the fraction of the IFN-γ–producing subset identified as NK cells (CD56+CD3−) versus T cells (CD3+CD56−) for PBMC samples from six unrelated healthy adults. (C) Freshly isolated PBMC samples exposed to plate-bound ICAM-1 were stained for CD3, CD8β, CD161, CD11a, IFN-γ, PLZF, and either CD1d tetramer, MR1 tetramer, or Vδ2 TCR. Results from three independent identically stained PBMC samples were concatenated, and the CD11ahi T cells were subjected to tSNE analysis. tSNE plots show the presence of IFN-γ+ cells in the iNKT cell (far left), MAIT cell (middle), and Vδ2+ T cell (right) subsets. Graph on far right shows the percent of the IFN-γ–producing population expressing these canonical TCRs for each of the PBMC donors that were included in the tSNE analyses. gMFI, geometric MFI.

Analysis of CD11ahi lymphocytes from PBMC samples stimulated by exposure to plate-bound ICAM-1–Fc revealed that IFN-γ–producing cells were distributed within the NK cell and T cell populations, with a trend toward colocalization with populations that coexpressed CD56 and CD161 and partial overlap with cells that expressed PLZF (Fig. 6B, left plots). Typically, the majority of the responding cells were T cells, with the remainder being NK cells (Fig. 6B, right plot). Using TCR-specific reagents, we observed that IFN-γ–producing cells were found within the iNKT cell, MAIT cell, and Vδ2+ T cell subsets (Fig. 6C). These results suggest that the ability to produce IFN-γ directly in response to a high surface density of ICAM-1 is a shared feature of human innate T lymphocytes and NK cells.

Discussion

The results presented in this paper delineate a novel pathway of integrin-mediated T cell activation. Prior studies have emphasized the ability of LFA-1 to contribute to T cell activation by lowering the threshold required for productive TCR signaling (34, 35). We show in this paper that innate T lymphocytes can directly use LFA-1 signaling to activate IFN-γ secretion. Because LFA-1 ligation occurs in the context of interactions with ICAM+ APCs, and LFA-1 engagement can enhance IFN-γ secretion in response to IL-12 stimulation, this activation pathway may facilitate the targeting of IFN-γ secretion toward specific types of recipient cells during inflammatory responses. Moreover, because IL-12 is not required, LFA-1–mediated activation may allow innate T cells to promulgate inflammatory responses independently of myeloid APCs in situations where an initiating event has led to the upregulation of ICAM on other cell types. For example, events such as the deposition of oxidized LDL or exposure to TNF-α or IL-1β are known to upregulate expression of ICAM-1 on vascular endothelial cells (61, 62). Because IFN-γ induces the upregulation of adhesion molecules on vascular endothelial cells, the integrin-mediated activation of iNKT cells in this context might play a role at the earliest stages of plaque formation by promoting the initial arrest of monocytic cells. Thus, we hypothesize that integrin-mediated activation of iNKT cells not only contributes to their responses during interactions with APCs that express CD1d and/or produce IL-12 but may also allow them to carry out functions in previously unrecognized situations where such APCs are not necessarily present.

One of the key features of the LFA-1–mediated activation pathway we observed is that it requires a threshold density of ICAM-1 to activate iNKT cell IFN-γ secretion. This may not be surprising, as it is known that LFA-1 binding to ICAM-1 involves a complex interplay between the conformational state of the LFA-1 molecule, which can adopt low-, intermediate-, or high-affinity states, and the avidity status induced by activation-induced clustering of LFA-1 at the cell surface (30). Thus, it is possible that the presence of a comparatively high density of ICAM-1 is sufficient to induce conformational changes in the LFA-1 molecules expressed by innate T lymphocytes that are associated with de novo signal transduction. It is intriguing to speculate that the high cell surface expression level of LFA-1 on innate T cells is thus a critical aspect of their ability to undergo independent LFA-mediated activation, as their elevated level of LFA-1 may enhance avidity-based interactions with ICAM.

We show in this paper that activation of iNKT cells by LFA-1 involves both Ca2+ and ERK signaling. We have previously demonstrated that human iNKT cells are highly responsive to weak TCR stimulation that generates ERK phosphorylation but only a minimal level of Ca2+ signaling (47). Moreover, we found that weak TCR engagement (even when it is not sufficient to directly induce a productive response) leads to acetylation of the IFNG locus of iNKT cells, and this epigenetic status renders them receptive to TCR-independent stimulation by IL-12 and IL-18 (46). It thus seems likely that the epigenetic status of the IFNG locus of innate T lymphocytes may also play a critical role in their ability to produce IFN-γ in response to LFA-1–mediated signals. Notably, although our studies suggest that CD1d recognition is not required for the activation of iNKT cells by high-density ICAM-1, it is not clear whether ICAM-1–mediated activation is likely to occur physiologically in the absence of TCR stimulation, because CD1d is present on a variety of cell types and the TCRs of human iNKT cells can bind with low-affinity to broadly distributed self-lipids (e.g., lyso-phosphatidylcholine).

However, our prior studies have suggested that human iNKT cells that are activated by weak TCR agonists (e.g., CD1d-presenting self Ags) produce mainly GM-CSF and not IFN-γ (47). In contrast, we noted that iNKT cell activation by high-density ICAM-1–Fc appears to selectively induce IFN-γ secretion, with little evidence of other cytokines typically produced by human iNKT cells, such as GM-CSF, IL-13, or IL-4 (data not shown). Thus, the presence of elevated ICAM-1 expression levels may serve to bias iNKT cell responses toward a Th1 phenotype even in the absence of strong TCR agonists or IL-12. Consistent with this, a prior report has also suggested that LFA-1 engagement promotes Th1 functions in iNKT cells (63).

This analysis outlines a previously unrecognized pathway of innate T cell activation that relies on signals resulting from LFA-1 binding to ICAM-1. It will be of significant interest in the future to determine whether this pathway allows innate T cells to influence other cell types in previously unrecognized situations. For example, naive T cells undergoing TCR-mediated activation have been shown to upregulate their cell surface ICAM-1 and form clusters with other T cells (64). LFA-1–induced IFN-γ secreted by iNKT cells in this context could thus promote Th1 polarization of naive T lymphocytes. Such interactions might contribute to the remarkable ability of iNKT cells to promote Ag-specific responses by adaptive T lymphocytes (65–70).

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Awards R01AI074940 and R01AI136500 to J.E.G.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CsA
    cyclosporine A
    α-GalCer
    α-galactosylceramide
    iNKT
    invariant NKT
    MAIT
    mucosal-associated invariant T
    MFI
    mean fluorescence intensity
    PLZF
    promyelocytic leukemia zinc finger
    tSNE
    t-distributed stochastic neighbor embedding.

  • Received April 16, 2018.
  • Accepted August 16, 2018.

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