CD2 promotes human natural killer cell membrane nanotube formation

Abstract

Membrane nanotubes are thin membranous projections that physically connect two cells. While nanotubes have been studied in human natural killer (NK) cells and are implicated in aiding NK cell cytotoxic function, requirements for their formation to susceptible target cells remain incompletely understood. Here we demonstrate that the CD2-CD58/48 receptor-ligand interaction promotes and is required for nanotube formation in human NK cells. In the CD2(-) NK cell line YTS, a stable CD2 expression variant enabled effective nanotube formation, and was associated with better cytotoxic function. Importantly, only interactions between an NK cell and a susceptible target cell were associated with multiple nanotubes and the number of nanotubes was inversely correlated with their length. Quantitative live cell fluorescence microscopy of CD2 nanotubes revealed time-dependent enrichment and localization of CD2 to the nanotube tip, and blocking CD2 receptor-ligand interactions prevented nanotube formation. Increased nanotube formation was not simply a feature of receptor-ligand pairing, as a KIR-MHC interaction in the same cell line system failed to promote nanotube formation. Additionally, blocking LFA-1-ICAM and 2B4-CD48 receptor-ligand interactions failed to inhibit nanotube formation. Thus only specific receptor-ligand pairs promote nanotubes. CD2 also promoted nanotube formation in ex vivo NK cells suggesting that CD2 plays a crucial role in the generation of nanotubes between an NK cell and its target.

Figure 1. YTS-CD2-GFP but not YTS-MyoIIA-GFP NK cells form stable NTs.

(A) The frequency of NT formation between NK cells and 721 cells was calculated as a percentage of NK cells forming NTs (black bars) and as a percentage of NK and 721 cell interactions leading to NT formation (white bars). See Materials and Methods for a detailed description of frequency calculations. YTS-MyoIIA-GFP cells were imaged with 721 cells on anti-CD48 coated plates, anti-CD48 and fibronectin (FN) coated plates, and on anti-CD48/FN coated plates with IL-2 pre-activation (n>100); YTS-CD2-GFP cells were imaged with 721 cells on anti-CD48 coated plates and on anti-CD48/FN coated plates (n>50). (B) YTS-MyoIIA-GFP NTs broke quickly, leaving thick projections ∼400 nm in diameter. (C) YTS-CD2-GFP NTs had a diameter of ∼100 nm and stably connected NK and target cells, sometimes for longer than 2 hours. (D) Cytotoxicity against 721 target cells is enhanced in YTS-CD2-GFP (black) cells in comparison to YTS (gray) cells (n = 3). (Scale bars: 5 µm.)

Figure 2. YTS-CD2-GFP cells can simultaneously form multiple NTs.

YTS-CD2-GFP cells were incubated with 721 cells for 30 minutes on anti-CD48 coated plates then imaged live using a spinning disc confocal microscope at a single time point. (A) Multiple NTs form between a single NK and target cell, or (B) connect an NK cell to multiple target cells. (C) The number of NK cells forming multiple NTs is inversely proportional to the number of NTs per cell (n>100 NTs). (D) As the number of NTs per cell increases, the average NT length decreases (1–3 NTs/Cell: n>25 NTs; 4 NTs/Cell: n>5 NTs). (Scale bars: 5 µm.)

Figure 3. CD2 localizes to the distal tip of the NK cell NT.

(A) A bright patch of CD2 fluorescence is seen at the distal tip of a YTS-CD2-GFP cell NT. Magnification of the boxed region depicts the NT tip becoming brighter over time as CD2 accumulates. (B) 65% of NK cell NTs exhibited an enrichment of CD2 at the NT tip as compared to the cell body. Enrichment persisted for the life of the NT (n>100 NTs). (C) Similarly, 85% of NK cell NTs exhibit enrichment of CD2 at the tip as compared to the overall length of the NT (n>100 NTs). (D), (E) and (F) Videos of NTs that show CD2 enrichment of the tip compared to the length of the NT were photo bleach-corrected, normalized to a unit time scale, time-binned, and averaged. Both sum (D) and mean fluorescent intensity (F) increased significantly over time, indicating that CD2 accumulates at the junction over time. (E) Tip area increased, but only marginally over time. Trend lines are shown as red dashed lines (n = 13 NTs). (Scale bars: 5 µm, Inset: 1 µm.)

Figure 4. YTS-CD2-GFP cells form more NTs than YTS-KIR-GFP cells.

(A) YTS-CD2-GFP and YTS-KIR-GFP cells were incubated with 721 cells for 30 minutes on anti-CD48 coated plates, then single images were obtained using live-cell confocal microscopy and scanning of z-planes to ensure NT detection. Bars show the percentage of NK cells exhibiting NK to target cell NTs (black) and NK to NK cell NTs (grey). See Materials and Methods section for a detailed description of frequency calculations. (B) Average NT length did not vary. Representative images of (C) YTS-KIR2DL1-GFP, with inset showing a lack of KIR enrichment at the tip and (D) YTS-CD2-GFP NTs. (n>320 cells) (Scale bars: 5 µm, Inset: 1 µm.)

Figure 5. CD2, but not KIR2DL1, LFA-1, or 2B4, receptor-ligand interactions promote NT formation.

(A) YTS-CD2-GFP cells were incubated with isotype or anti-CD2 blocking antibody for 15 minutes, followed by a 30 minute incubation with 721 target cells on anti-CD48 coated plates. Single time-point images were obtained to determine frequency of NT formation (n>350 cells). (B) YTS-KIR-GFP cells were incubated with 721-Cw4 cognate ligand-expressing cells or 721-Cw3 non-cognate ligand-expressing cells on anti-CD48 coated plates for 30 minutes. The frequency of YTS-CD2-GFP cells incubated with 721 cells (from Fig. 4A) is included for comparison (n≥300 cells). (C) YTS-CD2-GFP cells were incubated with isotype or anti-LFA-1 blocking antibody for 15 minutes, followed by a 30 minute incubation with 721 target cells on anti-CD48 coated plates. Single time-point images were obtained to determine frequency of NT formation (n>500 cells). (D) YTS-CD2-GFP cells were incubated with isotype, anti-CD2 blocking antibody, or anti-2B4 blocking antibody for 15 minutes, followed by a 30 minute incubation with 721 target cells on anti-CD20 and FN coated plates. Single time-point images were obtained to determine frequency of NT formation (n>500 cells). Bars show the percentage of NK cells exhibiting NK to target cell NTs (black) and NK to NK cell NTs (grey).

Figure 6. CD2 receptor-ligand interactions promote NT formation in eNK cells.

(A) NTs were visualized in PKH26 labeled eNK cells isolated from whole blood. (B) eNK cells were labeled with PKH26, treated with isotype or anti-CD2 blocking antibody for 15 minutes, then incubated with 721 cells on anti-CD48 coated plates for 30 minutes. Single images were obtained to determine NT frequency (n>850 cells). (C) eNK cells were sorted into CD2⁺ and CD2⁻ populations using a non-blocking antibody to CD2. (D) CD2⁺ and CD2⁻ eNK populations were labeled with PKH26, and single images were obtained to determine the frequency of NK-NK (black bar) and NK-TC (grey bar) NT formation (n>325 cells). (Scale bar: 5 µm.).