Immunosynapse formation coincides with rapid activation of NK cells by syngeneic T cells and correlates with clustering of MHC class I
Brian A. Rabinovich1,
Jennifer Li2,3,
Rose Hurren3 and
Richard G. Miller2,3,4
1 Amgen Washington, 1201 Amgen Court West, Seattle, WA 98119, USA
2 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
3 Ontario Cancer Institute, Division of Cell and Molecular Biology, Room 9-305, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada
4 Department of Immunology, University of Toronto, Toronto, Ontario, Canada
Correspondence to: R. G. Miller; E-mail: miller{at}oci.utoronto.ca
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Abstract
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T cells cultured for 3 h with antigen-presenting cells (APCs) stimulated syngeneic IL-2-activated NK cells as measured via a standard chromium-release assay. Discrete caps containing both TCR and MHC-I had formed on the surface of these activated T cells. When conjugates were formed between NK cells and these activated T cells, >80% of the contact sites were in the MHC-Idim region outside the TCRMHC-I cap. Stimulation with phorbol myristate acetate plus Ionomycin, which bypasses the need for cell surface events during activation, did not induce either cap formation or NK cell activation. Further, the addition of the protein transport inhibitor Brefeldin A did not block activation of NK cells. MHC-I is the major inhibitory ligand recognized by NK cells. One possible mechanism for the activation of NK cells by TCRMHC-I-capped T cells is that aggregation of MHC-I into one region leaves the remaining T cell surface denuded of ligands for NK-inhibitory receptors. As a test of this hypothesis, we aggregated MHC-I on T cells with plate-bound anti-MHC-I mAb. This treatment conferred upon the T cells the capacity to activate NK cells, suggesting that MHC-I clustering could contribute to the observed phenomenon.
Keywords: cytotoxicity, immunological synapse, lymphokine-activated killer cells, mice, T-lymphocytes
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Introduction
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MHC-I plays a critical role in the protection of cells, including lymphocytes, from NK (1, 2). Several families of NK cell-inhibitory receptors are specific for MHC-I. These include Ly49 (mice) (3, 4), CD94 (mice and humans) (5, 6) and the killer-inhibitory receptor (humans) (4, 7). The Missing Self hypothesis, envisioned by Kärre in 1986, was the first model to correctly predict that all NK cells express at least one inhibitory receptor specific for self-MHC-I (8, 9). Thus, when MHC-I is depressed or absent, NK cell activation can occur.
During the course of T cell activation by an antigen-presenting cell (APC), an immunological synapse containing clusters of TCR and MHC forms between T cells and APCs. This synapse is composed of cholesterol-dense lipid rafts, forms within minutes of TAPC engagement and is necessary for full T cell activation, including proliferation and protein synthesis (10, 11). Previously, we reported that T cells transport ligands for NK cell activation receptors (e.g. NKG2D) to their cell surfaces within 15 h of activation. This results in the formation of a highly sensitive NK cell target (12). Here we report that activated T cells are already moderately sensitive to lysis by a 3 h post-activation and provide evidence that sensitivity is associated with the re-distribution of cell surface molecules, perhaps most importantly MHC-I, and not protein transport to the cell surface.
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Methods
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Animals
C57BL/6 (B6) and BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). B6 and BALB/c FcR
knockout mice were purchased from Taconic Labs (Germantown, NY, USA). D011.10 C.B-17 SCID TCR trangenic mice specific for amino acid residues 323339 of chicken ovalbumin peptide (OVA; in the context of H-2I-Ad) (13) were engineered at the Ontario Cancer Institute (Toronto, Canada) by breeding TCR transgenic mice (Jackson Laboratory) (13) onto the C.B-17 (congenic to BALB/c except for IgH locus) SCID background. 2C TCR transgenic mice specific for H-2Ld, presenting the ubiquitous mitochondrial peptide, p2Ca (14, 15), were obtained from D. Y. Loh (16). H2-Db-deficient mice on the C57BL/6 background expressed only the H2-Kb MHC-I molecule as previously described (17). Mice were kept in a specific pathogen-free animal colony in the Ontario Cancer Institute. In most experiments, 6- to 8-week old mice were used.
Reagents
Hybridomas 2.4G2 (rat anti-mouse Fc
RIII-
) and 1B2 (rat anti-mouse 2C TCR) were obtained from the American Type Tissue Collection (ATCC, Rockville, MD, USA) and mAbs were purified from tissue culture supernatants as previously described (18). The following mAbs were purchased from Pharmingen (San Diego, CA, USA): purified mouse anti-DNP and fluorescently labeled anti-NK1.1 (PK136), anti-H2-Kb, anti-H-2Db and anti-TCRß. Labeling of purified antibodies with FITC was performed as previously described (18). Antibodies were tagged with Cy3 using a Cy3-labeling kit from Amersham (Piscataway, NJ, USA). Brefeldin A (BFA), Con A, phorbol myristate acetate (PMA) and Ionomycin were purchased from Sigma Chemical Co. (St Louis, MO, USA). Flow cytometric acquisition was performed using a Becton Dickinson FACSCalibur and analyzed with FCS Express software (Novosoft, Toronto, Ontario, Canada).
T cell activation
T cells were isolated from splenocyte suspensions via the removal of B cells with anti-mouse B220 Dynabeads (Dynal, Oslo, Norway) and cultured in complete medium (CM) with other additives as indicated. CM consisted of
-MEM (Life Technologies, Burlington, Ontario, Canada) supplemented with 10% FCS (Life Technologies), 50 µM 2-mercaptoethanol and 10 mM HEPES buffer. For activation of 2C T cells, 23 x 106 T cells were suspended in 5 ml CM containing 515 mouse units of human rIL-2 per milliliter and plated onto BALB/c or F1 splenic adherent cells irradiated with 20 Gy
-radiation in one well of a six-well plate. Plates were spun for 3 min at 700 revolutions per minute (r.p.m.) and incubated for various times as indicated at 37°C, 7% CO2. If APCs were allogeneic to the NK cells used in the killing assay, they were removed after activation as follows: cells were stained with mAbs anti-H2-Dd (HB87, ATCC) and anti-H2-Kd (K9.18.10s; kind gift of W. Jefferies, University of British Columbia, Vancouver, British Columbia, Canada), washed and removed with anti-mouse IgG Dynabeads (Dynal). For activation of DO.11.10 T cells, BALB/c plastic adherent spleen cells were first pulsed overnight with 1 mg ml1 chicken OVA before addition of T cells in the same manner as described above for 2C. Activation of T cells with Con A was performed as previously described (18) and the cells were washed with 200 mM
-methyl-mannoside
-methyl-glucoside prior to the cytotoxicity assay. For activation with PMA and Ionomycin, 23 x 106 T cells were suspended in 5 ml CM plus 10 ng ml1 PMA and 250 ng ml1 Ionomycin in one well of a six-well plate and incubated for various times at 37°C, 7% CO2.
Cytotoxicity assay
Lymphokine-activated killer cells were generated as previously described (18). Chromium-release assays using T cells as 51Cr-labeled targets were performed as previously described (18). The use of BFA to disrupt the Golgi apparatus in target cells without affecting NK cell effector function was performed as previously described (19). Briefly, T cells were first incubated in a high concentration of BFA (5 µg ml1 for 45 min), a dose sufficient to disrupt the Golgi. Cells were then activated and assayed in the presence of 0.5 µg ml1 BFA. This lower concentration prevents the Golgi from re-forming in cells pre-exposed to 5 µg ml1 BFA but does not affect the ability of NK cells to kill. For chromium-release assays involving immobilized mAbs, anti-H2-Kb or anti-DNP (Pharmingen) was bound to 96-well round-bottom plates in a 1-µl volume at 0.5 µg per well overnight at 4°C. The plates were subsequently washed three times before the addition of chromium-labeled targets at 6000 cells per well, also in 1 µl, and pre-treated with 5 µg ml1 BFA. After a 30-min incubation to induce H2-Kb clustering, 100 µl CM containing 1 µg ml1 BFA was added. NK effectors were then added in 100 µl as above at the indicated E : T, the plates were then spun for 5 min at 800 r.p.m. and incubated for 4 h at 37°C, 7% CO2.
Immunofluorescence and confocal microscopy
Immunofluorescence microscopy and/or confocal microscopy were used to visualize the distribution of TCR and H2-Db on resting and activated T cells. Briefly, T cells were stained with hamster anti-mouse TCR-FITC (Pharmingen) and/or Cy3-labeled anti-mouse H2-Db (Pharmingen) in the presence of 0.1% NaN3 for 30 min on ice. Cells were subsequently washed, fixed with 2% PFA and spun onto Ultrafrost Plus slides (VWR) at 1200 r.p.m. using plate carriers modified to hold standard microscope slides. Slides were subsequently mounted in Vectamount with DAPI (Vector Labs), and fluorescence was visualized using a Leica fluorescent microscope or Zeiss confocal microscope. Images were captured using a Photometrics Snappy digital camera and software or Zeiss LSM 510 Image Browser, respectively.
For immunofluorescence visualization of TNK conjugates, 1 x 106 purified T cells were first stained with hamster anti-mouse TCR-FITC (Pharmingen) and lightly fixed with 2% PFA for 510 min at room temperature to prevent endocytosis of antibodies during the subsequent conjugate formation step. One million syngeneic NK cells were labeled with 25 µM Cell-tracker Blue in serum-free
-MEM for 4560 min, washed and mixed with T cells in CM. Mixtures were spun for 5 min at 1200 r.p.m. and incubated for 20 min at 37°C. The conjugates were then processed and visualized as described above.
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Results
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T cells stimulated for 3 h by APC-activated NK cells
We found that, upon interaction with APCs, both CD4+ and CD8+ T-lymphocytes gain the capacity to stimulate IL-2-activated NK cells (referred to as NK cells). We used standard chromium-release assays as our readout. CB-17 CD4+ Ova transgenic T cells (D011) were activated for varying times on BALB/c APCs pulsed with OVA. Figure 1(A) shows that a substantial level of lysis could be seen at both 3 and 6 h, and
2.5-fold higher level of lysis at 15 h. When B6 CD8+ 2C trangenic T cells specific for H-2Ld were activated on BALB/c APCs, we found a similar two-phase response (Fig. 1B). These data indicate that T cells co-cultured with APCs for 36 h have the capacity to stimulate NK cells. To determine whether NK cell activation required presentation of T-specific antigen, we deliberately activated D011 or 2C trangenic T cells on APCs not pulsed with OVA or missing H-2Ld (i.e. dm2), respectively. Figure 1(C) shows that 2C T cells activated on dm2 APCs could not stimulate B6 NK cells, even after 6 h of co-culture. Similar results were obtained with D011 T cells incubated on APCs without OVA (data not shown).

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Fig. 1. Following activation, T cells are rendered sensitive to syngeneic NK cell-mediated lysis. (A) CD4+ D011 T cells were activated on BALB/c APCs pulsed with chicken OVA for 0 (circles), 3 (squares), 6 (triangles) or 15 h (diamonds). T cells were subsequently tested for sensitivity to day 4 BALB/c NK cells in a 4-h chromium-release assay. Specific lysis values at E : T from 3 : 1 to 30 : 1 are shown. (B) CD8+ 2C T cells were activated on BALB/c APCs for 0 (filled circles), 3 (filled squares), 6 (filled triangles) and 15 h (filled diamonds) and assayed for sensitivity to day 4 B6 NK cells as in (A). Specific lysis values at E : T from 3 : 1 to 30 : 1 are shown. (C) 2C T cells were activated as in (B) for 0 (filled circles) or 6 h (filled squares) or for 6 h on dm2 (Ld) APCs (open squares). Cells were harvested at these time points and used as targets for B6 NK cells in a chromium-release assay as in (B). Specific lysis values at E : T from 3 : 1 to 30 : 1 are shown. (D) 2C T cells were activated for 6 or 20 h as in (B) with or without BFA as indicated and tested for sensitivity to B6 NK cells. Resting T cells are shown as black bars and activated T cells as white bars. Specific lysis values at an E : T of 30 : 1 are shown.
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Next, we tested whether T cell-mediated NK cell activation required export of proteins to the T cell surface. BFA, a Golgi-disrupting agent, was used to inhibit protein transport in a manner that restricted its activity to 2C T cells so that neither APC nor NK cell was affected (19) as described in Methods. The ability of T cells to stimulate NK cells during this early phase remained intact. However, as previously reported, late-phase activation was strongly inhibited (Fig. 1D) (12). Similar results were observed with D011 T cells (data not shown).
To test whether an activation process that bypasses surface interactions between APCs and T cells could lead to NK activation, we stimulated T cells with PMA plus Ionomycin, which directly signals to protein kinase C (PKC) and mobilizes intracellular calcium (20). We activated 2C T cells on APCs or with PMA plus Ionomycin for 3 and 20 h. Only 2C T cells co-cultured with APCs activated NK cells at the 3-h time point (Table 1). In contrast, both PMA plus Ionomycin and APCs induced comparable NK cell activation at 20 h. Similar results were obtained for D011 T cells (data not shown).
NK cell activation is associated with TCR capping and TCRMHC-I co-clustering
Our data suggest that the export of new proteins to the cell surface is not required for NK cell stimulation mediated by newly activated T cells. Since activation at 3 h was completely dependent on co-culture with APCs, it is possible that the re-arrangement of materials already on the cell surface may contribute to the capacity of these T cells to activate NK cells. Given that T cells form an immunological synapse with cognate APCs that involves clustering of TCR and MHC-I, we decided to test this hypothesis. We stained T cells with a FITC-labeled anti-TCR mAb and looked for TCR capping by confocal microscopy. At 36 h after activation of 2C T cells on APCs, we observed that 84% of cells contained TCR caps similar to that shown in Fig. 2(C). Cap formation clearly resulted in a non-uniform distribution of surface proteins. We examined whether the contact points between TCR-capped T cells and NK cells were random or had a defined relationship with respect to the cap. B6 FcR
/ NK cells were used to avoid antibody-dependent conjugation between NK cells and mAb-coated 2C targets (21). 2C T cells were activated for 6 h on APCs, their TCR tagged with a green fluorescent antibody and conjugates formed between Cell-tracker Blue-labeled NK cells and T cells. The contact point in most TNK cell conjugates appeared to be outside the T cell cap. Figure 2(D) is a representative example. To quantify this impression, 200 conjugates between NK cells (blue) and T cells (green) were scored sequentially for binding location: of these, 200, 168 or 84% made central contact entirely outside the TCR cap. The remaining 32 appeared to make contact in both the TCRbright and TCRdim regions.

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Fig. 2. NK cells bind to capped T cells in a contact site outside the cap containing both TCR and MHC-I. On day 4 H-2Db/ B6 NK cells (pre-coated with anti-FcRIII Fab) were labeled with the blue fluorescent dye, Cell-tracker Blue (blue, panel B). Conjugates were then formed as described in Methods with 2C T cells activated on BALB/c APCs for 8 h (panel D). 2C T cells were stained with anti-H-2DbCy3 (red, panel A) and 1B2 anti-TCRFITC (green, panel C). Panel D shows a combined image of all three colors.
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MHC-I molecules are important NK cell-inhibitory ligands. Therefore, we stained capped 2C T cells for TCR and H-2Db (MHC-I) simultaneously (Fig. 2A and C). First, we found that MHC-I co-localized in the cap with the TCR. Second, by forming conjugates between these double fluorescently tagged T cells and Cell-tracker Blue-labeled NK cells, we noted that the TNK cell contact point was in the MHC-Idim region (Fig. 2D). Masking of H-2Db on T cells results in the activation of NK cells bearing inhibitory receptors for H-2Db. We therefore used NK cells from B6 H2-Db/ mice, which have developed in an environment devoid of selfDb and thus do not respond to its absence. Similar results were observed with wild-type TNK cell conjugates (data not shown).
Artificial clustering of MHC-I on resistant T cells results in rapid sensitization
Perhaps NK cells are activated by capped T cells because upon binding to the MHC-Idim region, there is insufficient inhibitory signal to block activation. It is formally possible that capping of MHC-I is sufficient to induce such activation. To test this hypothesis, we clustered H-2Kb on 51Cr-labeled H-2Db/ T blasts using plate-immobilized anti-H-2Kb mAb. T cells blasted for 3 days with Con A were used because they are known to be resistant to lysis by syngeneic NK cells (12). Once the T cells were bound to the plate, exposing their MHC-Idim surface, we added syngeneic H-2Db/ NK cells, thus permitting the study of a single inhibitory receptorligand interaction (19). As seen in Fig. 3, a 2-fold increase in sensitivity to lysis was observed when the T cells were bound to immobilized anti-H-2Kb as compared with immobilized anti-DNP. To prevent the de novo transport of proteins onto the exposed surface of T cells during the chromium-release assay, BFA was used as described in Methods. When BFA was omitted, sensitization did not occur, indicating that free anti-MHC-I mAb did not contaminate the assay wells, which would have blocked the interaction between newly transported MHC-I and NK-inhibitory receptors (data not shown).

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Fig. 3. Clustering of MHC-I with immobilized anti-H2-Kb antibody on NK cell-resistant T blasts results in conversion to a sensitive phenotype. H-2Db/ T cells were activated for 3.5 days with Con A (2 µg ml1), labeled with 51Cr and treated with 5 µg ml1 BFA to prevent de novo transport of MHC-I to the cell membrane. The cells were then plated into 96-well round-bottom plates coated with PBS only (clear bar), immobilized anti-H2-Kb antibody (black bar) or immobilized anti-DNP antibody (gray bar) and their sensitivity to H-2Db/ NK cell-mediated lysis was measured at an E : T of 20 : 1.
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Discussion
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We have shown previously that by 15 h post-activation, T cells gain the capacity to vigorously stimulate NK cells. This results from the export of ligands for NK activation receptors (e.g. NKG2D) to the T cell plasma membrane. We refer to these events at 15 h as the late phase. Since the late phase was dependent on the up-regulation of cell surface proteins, it was not surprising that BFA was inhibitory. Further, NK cell activation was mediated by T cells stimulated not only by APCs, but also by PMAIonomycin or Con A, the latter two of which activate T cells downstream of cell surface events (12).
In this report, we found that T cells have already gained some capacity to activate NK cells within 3 h of activation in a process that does not appear to require the appearance of new ligands on the cell surface. Importantly, APCs but not PMA plus Ionomycin could induce this early phase of T cell-mediated NK cell activation. When APCs were used to activate T cells, >84% of T cells contained TCR caps at 36 h. In contrast, treatment with PMA plus Ionomycin, which does not ligate the TCR but rather activates intracellular PKC (PMA) and mobilizes calcium (Ionomycin) (20), did not induce TCR capping at these times (data not shown) and did not induce an early-phase response. This suggested that the APC-induced re-orientation of pre-existing molecules on the T cell surface conferred upon the T cell the capacity to activate NK cells. Consistent with our hypothesis, we found that molecules on the T cell surface had been re-distributed into TCRMHC-Ihigh and TCRMHC-Ilow microdomains and that the latter was the surface interacting with NK cells. We found that the TCR cap was a relatively stable structure, which dissociated over a 14-h period by first breaking up into a patch network. This explains why we were able to measure sensitivity without APCs present in the cytotoxicity assay. Further, we did not observe decreased levels of total MHC-I on activated T cells relative to resting T cells (data not shown). We speculate that clustering of protective MHC-I may leave a region outside the cap relatively devoid of inhibitory ligands. This region may be suitable for NK cell conjugation concomitant with a decreased threshold for activation. Although we limited our study to the measurement of MHC-I, it is formally possible that other inhibitory receptorligand pairs such as CD94Qa-1 are also involved (22).
Based on our observation that co-culturing T cells with APCs results in a discrete MHC-Ilow region, it is possible that MHC-I clustering is sufficient to confer the ability to stimulate NK cells. We thus clustered MHC-I on T cells using immobilized anti-H2-Kb. We wanted to avoid separating NK cells into subsets expressing inhibitory receptors for Kb and Db as those for the latter have not yet been identified. Thus, we used a system in which only Ly49C and H2-Kb should contribute to NK cell inhibition as it has been shown that Ly49C is the only Ly49-inhibitory receptor with specificity for H-2Kb (19). We found that T cells bound to immobilized anti-H2-Kb were capable of activating NK cells. This suggests that isolated clustering of MHC-I is sufficient for induction of sensitivity. When the experiment was repeated using NK cells fractionated into Ly49C+ and Ly49C subsets, we found that Ly49C NK cells were also slightly activated, albeit less than their Ly49C+ counterparts (data not shown). This implies that a receptorligand interaction other than Ly49C/Kb is both affected by MHC-I clustering and involved in regulating NK activation. A caveat to this whole approach is that it has been recently shown that NK cells educated in the absence of cognate-inhibitory ligands are functionally impaired (23), thus raising questions as to the physiological relevance of any data obtained with the Ly49C H2-Db/ NK cells. We would speculate, however, that CD94Qa-1 interactions may play a role in this system.
Our study does not offer an explanation for the mechanism by which MHC-I co-clusters with the TCR within the T cell cap. One possibility is that a substrate for MHC-I on the APCs pulls T cell-derived MHC-I into the cap. This is especially attractive because it might suggest that such a molecule could be involved in dislodging the APCs from the T cells after sufficient activation has occurred. A candidate might be the Leukocyte Ig-like Receptor (LIR)-like, Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-containing receptor, Paired Ig-like Receptor B (PIR-B) which was very recently reported to bind MHC-I (24).
In conclusion, two phases of T cell-mediated NK cell activation can now be resolved. The early phase at 36 h appears to be the result of cell surface re-organization which effectively removes an inhibitory ligand from part of the cell surface, perhaps MHC-I, while the late phase results from up-regulation of ligands for NK cell activation receptors (12). We speculate that both mechanisms may play a role in immunoregulation. Depending on the cytolytic status of the NK cell, activation may lead to either cytokine production or cytotoxicity.
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Acknowledgements
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This work was supported by a research grant to R.G.M. from the National Cancer Institute of Canada and funds to B.A.R. from the Ontario Graduate Student Program.
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Abbreviations
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APC | antigen-presenting cell |
ATCC | American Type Tissue Collection |
BFA | Brefeldin A |
CM | complete medium |
OVA | ovalbumin peptide |
PKC | protein kinase C |
PMA | phorbol myristate acetate |
r.p.m. | revolutions per minute |
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Notes
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Transmitting editor: W. M. Yokoyama
Received 14 January 2004,
accepted 1 March 2005.
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