Requirement of Species-Specific Interactions for the Activation of Human γδ T Cells by Pamidronate

Materials and Methods

Results

Requirement of LFA-1/ICAM-1-mediated adhesive interaction for effective activation of human γδ T cells by pamidronate-pulsed human tumor cells

We previously reported that pamidronate specifically induced the clustering between human γδ T cells and accessory monocytes, suggesting the requirement of direct cellular adhesion for effective γδ T cell activation (9). As indicated in Fig. 1⇓A, both primary and activated human γδ T cells highly expressed LFA-1 in an active form. On the other hand, human tumor cell lines, which could efficiently present pamidronate to γδ T cells, expressed ICAM-1, with representative examples being indicated in Fig. 1⇓B. In contrast, a minor population of human tumor cell lines with no or only weak capacity to activate γδ T cells exhibited only marginal LFA-1 ligand expression including ICAM-1, -2, and -3 (Fig. 1⇓B). Although not shown, ICAM expression levels on tumor cells were not affected by pamidronate-pulsing. γδ T cells and ICAM-1^high EJ-1 tumor cells either mock treated or pulsed with pamidronate formed significant cellular conjugates by 30 min after the mixture (Fig. 2⇓A). The conjugate formation with untreated EJ-1 cells was only transient and dissolved nearly to the background level in 2 h. On the other hand, the cellular conjugates between γδ T cells and pamidronate-pulsed EJ-1 cells lasted stably at least for 2 h (Fig. 2⇓A). Inclusion of a blocking anti-LFA-1 mAb in the cocultures of γδ T cells and pamidronate-pulsed EJ-1 cells significantly inhibited the conjugate formation and subsequent IFN-γ production as well (Fig. 2⇓B). These results strongly suggested that LFA-1/ICAM-1-mediated adhesive interaction with pamidronate-pulsed human tumor cells was one of the requirements for effective activation of γδ T cells.

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FIGURE 1.

Expression of LFA-1 on human γδ T cells and ICAMs on human tumor cell lines. A, Two-color staining of freshly isolated normal PBMC with anti-TCR Vδ2 and anti-LFA-1 mAb (upper) or NKI-L16 mAb specific for an activated form of LFA-1 (lower). B, Staining of representative human tumor cell lines capable of strongly activating human γδ T cells with pamidronate (EJ-1, VMRC-RCW, and K562) and those with poor or no activity to stimulate γδ T cells (LK-2 and MKN45) with anti-ICAM-1, -2, and -3 (bold lines). Fine lines indicate control staining.

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FIGURE 2.

LFA-1-dependent conjugate formation between human γδ T cells and pamidronate-pulsed EJ-1 tumor cells and γδ T cell activation. A, CFSE-labeled γδ T cells and PKH26-labeled EJ-1 tumor cells either unpulsed (○) or pulsed with 0.1 mM pamidronate (•) were mixed at 37°C for indicated periods and were two-color analyzed by FACScan. Percent conjugated cells represent the percentage of double-positive γδ T cells divided by total γδ T cells. A bar indicates background value of the mixture with no prior incubation. B, Similarly labeled γδ T cells and EJ-1 tumor cells pulsed with 0.1 mM pamidronate were incubated at 37°C for 2 h in the absence (□) or presence (▪) of anti-LFA-1 Ab (40 μg/ml), and percent conjugated cells were determined as above. A bar indicates background value of the mixture with no prior incubation. The experiments were repeated twice with identical results. C, γδ T cells and EJ-1 tumor cells mock treated (left) or pulsed with 0.1 mM pamidronate were incubated at 37°C for 2 h in the absence (middle) or presence (right) of anti-LFA-1 Ab (40 μg/ml), and IFN-γ-producing cells were determined by two-color staining. Proportions of IFN-γ-producing γδ T cells are indicated.

LFA-1/ICAM-mediated adhesive interaction is necessary but not sufficient for γδ T cell activation by pamidronate-pulsed human tumor cells

These results raised a possibility that failure of γδ T cell activation by a minor population of human tumor cell lines pulsed with pamidronate was due to the insufficient expression of ICAM-1 on the cell surface. To examine this, we transfected a human ICAM-1 cDNA into ICAM-1^low LK-2 and MKN45 cells, which induced only weak or no detectable activation in γδ T cells (see Fig. 1⇑B and Table I⇑), and established ICAM-1^high clones (Fig. 3⇓Aa). A pamidronate-pulsed ICAM-1^high LK-2 clone induced a much stronger activation of γδ T cells than wild-type cells in terms of both IFN-γ production and specific proliferation (Fig. 3⇓A, b-d). The results confirmed that inefficient activation of γδ T cells by pamidronate-pulsed LK-2 tumor cells was primarily due to the low expression of ICAM-1. In quite a contrast, an ICAM-1^high MKN45 clone pulsed with pamidronate remained defective in activating γδ T cells (Fig. 3⇓B). Essentially the same results were obtained by using other independent ICAM-1^high MKN45 clones. Thus, it was suggested that LFA-1/ICAM-mediated interaction was required but not sufficient for the effective activation of human γδ T cells by tumor cells pulsed with pamidronate.

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FIGURE 3.

Effect of ICAM-1 cDNA transfection into the ICAM-1^low human tumor cell lines on the capacity to activate γδ T cells upon treatment with pamidronate. LK-2 (A) and MKN45 (B) tumor cell lines were transfected with ICAM-1 cDNA and analyzed for ICAM-1 expression (a). Dotted lines, control; solid lines, wild-type tumor cells stained with anti-ICAM-1; and bold line, transfected clones stained with anti-ICAM-1 mAb. Human γδ T cells were incubated with the transfectants mock treated (b) or pulsed with 0.1 mM pamidronate (c) for 4 h, and IFN-γ-producing cells were determined. Human γδ T cells (10⁵ cells/well) were cocultured with varying numbers of wild-type tumor cells (○) or ICAM-1 transfectants (•) pulsed with 0.1 mM pamidronate for 2 days, and the proliferation was assessed. The means and SE of triplicate cultures are indicated.

Expression of human ICAM-1 does not confer the capacity of activating human γδ T cells to murine tumor cell lines

We finally examined the effect of human ICAM-1 expression on murine tumor cell lines. A mouse B16 melanoma clone transfected with human ICAM-1 cDNA (hICAM-1^high B16) expressed a high level of human ICAM-1 comparable to that of EJ-1 human tumor cells (Fig. 4⇓A). However, hICAM-1^high B16 cells pulsed with pamidronate failed to activate human γδ T cells with respect to either IFN-γ production or specific proliferation (Fig. 4⇓, B and C). In addition, human ICAM-1^high B16 cells pulsed with pamidronate remained totally resistant to cytotoxicity elicited by human γδ T cells (data not shown). Identical results were obtained using hICAM-1^high P3U1 mouse myeloma clones (data not shown). These results strongly suggested that the lack of human γδ T cell activation by pamidronate-pulsed tumor cells of nonhuman origins was not only due to the defective LFA-1/ICAM-1-mediated interaction with human γδ T cells, but also to the absence of additional human-specific element(s) for the effective activation.

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FIGURE 4.

Effect of human ICAM-1 cDNA transfection into a mouse tumor cell line on the activation of human γδ T cells by the treatment with pamidronate. The murine B16 melanoma cell line was transfected with human ICAM-1 cDNA. A, Expression of human ICAM-1. Dotted lines, control; solid lines, wild-type tumor cells stained with anti-hICAM-1; and bold line, transfected clone stained with anti-hICAM-1. B, Human γδ T cells were incubated with transfectants untreated (left) or pulsed with 0.1 mM pamidronate (right) for 4 h, and IFN-γ-producing cells were determined. C, Human γδ T cells (10⁵ cells/well) were cocultured with varying numbers of the wild-type tumor cells (○) or ICAM-1 transfectants (•) pulsed with 0.1 mM pamidronate for 2 days, and the proliferation was assayed.

Discussion

Human γδ T cells bearing Vγ2Jγ1.2Vδ2-TCR recognize several kinds of small nonpeptide Ags with distinct chemical features (1, 10), but the exact mode of Ag recognition remains unknown. Previous reports indicated that homologous aggregation among γδ T cells was required for their activation by some of the nonpeptide Ags (14). More recently, it was reported also that human γδ T cells formed stable conjugation with the susceptible tumor target cells with synaptic molecular transfer resembling immunological synapse between αβ T cells and specific APC (15). Although these results implied that cell-associated presentation of nonpeptide Ags might be required for γδ T cell activation, detailed analysis was hampered by the fact that most of the nonpeptide Ags could efficiently activate γδ T cells even in the absence of other specific cells (14). In this aspect, we indicated that the primary human γδ T cells failed to respond to soluble pamidronate, but recognized the compound in the presence of accessory monocytes (9). In addition, we also showed that many human tumor cells prepulsed with pamidronate could efficiently activate γδ T cells (13). Such a requirement of Ag presentation was a common feature of all of the antigenic nitrogen-containing bisphosphonate molecules (S. Kita, Y. Tanaka, and N. Minato, unpublished observation), which might provide an ideal system for detailed analysis of γδ T cell activation by nonpeptide Ags.

The present results indicated that the majority of pamidronate-pulsed human tumor cell lines of varying tissue origins (21of 26 lines) activated γδ T cells, whereas none of the tumor cell lines of nonhuman origins could activate human γδ T cells (0 of 33 lines). After the pulsing procedure, no significant toxicity was detected in all of the tumor cell lines of both human and nonhuman origins as judged by propidium iodide staining. Thus, it was unlikely that γδ T cells were activated simply by pamidronate nonspecifically bound to the target tumor cells. Incidentally, the present results also suggested that most human Vγ2Vδ2 γδ T cells could not recognize xenogeneic MHC Ags, while human αβ T cells were shown to recognize xenogenic MHC Ags directly and exhibit comparable levels of responses to allogeneic cells (16).

Human γδ T cells highly expressed LFA-1 molecules on their cell surface, and ICAM-1 was detected on most of the human tumor cell lines that could present pamidronate. Then, γδ T cells formed tight conjugates with the pamidronate-pulsed tumor cells. Inclusion of a blocking anti-LFA-1 mAb in the cocultures significantly inhibited the conjugate formation, resulting in the inhibition of IFN-γ production by γδ T cells. The results suggested that LFA-1/ICAM-1-mediated adhesive interaction was required for effective γδ T cell activation. Consistently, ICAM-1^low LK-2 lung tumor cell line gained a strong capacity of activating γδ T cells by ICAM-1 cDNA transfection upon pretreatment with pamidronate. Although cell clusters between γδ T cells and mock-treated tumor cells were only transient, γδ T cells formed stable conjugates with pamidronate-pulsed tumor cells, lasting for at least 2 h. In αβ T cells, it is indicated that LFA-1 is activated and reorganized to form stable immunological synapse with APC via “inside-out” signaling mediated by TCR (17, 18, 19, 20). In analogy, we speculate that the γδ TCR signaling via cell-associated pamidronate may induce reorganization of LFA-1 to form stable synapse with the Ag-presenting tumor cells.

The present results further indicated that the integrin-mediated interaction was not sufficient for pamidronate-pulsed tumor cells to activate human γδ T cells. Thus, a high level of ICAM-1 expression by cDNA transfection failed to confer the capacity of γδ T cell activation to the ICAM-1^low MKN45 human tumor cell line. Furthermore, expression of human ICAM-1 in murine tumor cell lines could not induce the capacity to activate human γδ T cells at all. These results strongly suggested that additional specific interaction other than integrin-mediated adhesion was required for γδ T cell activation by pamidronate-pulsed tumor cells.

Although the exact nature of interaction between γδ T cells and pamidronate-presenting cells remains to be investigated, at least two possible models are considered, which are not exclusive to each other. First, although the γδ TCR may directly interact with the native nonpeptide Ags presented on the target cells, additional costimulatory signals via specific ligands may be needed for the efficient γδ TCR signaling like the B7 family molecules in the case of αβ T cell activation (21, 22). Although human γδ T cells express a significant level of CD28 (our unpublished data), few tumor cell lines exhibit B7-1 or B7-2 expression, and the candidate molecules remain to be identified. Alternatively, γδ TCR may recognize nitrogen-containing bisphosphonate Ags only in association with specific molecules on the Ag-presenting tumor cells. Our unpublished results indicated that, in addition to the Jγ1.2-encoded lysine residues (11), at least two more additional residues in the putative Ag-binding pocket in Vγ2Jγ1.2Vδ2 TCR were required for γδ T cell activation by diverse nonpeptide Ags (S. Yamashita, Y. Tanaka, and N. Minato, manuscript in preparation). Thus, it is possible that nonpeptide Ags along with specific Ag-presenting molecules optimally fit to the pocket via anchoring residues in much the same way as peptide Ags in complex with MHC molecules for αβ TCR. A number of previous studies provided no evidence for involvement of MHC in γδ T cell activation by nonpeptide Ags (14, 23, 24) and again the candidate molecules remain to be seen.

The present results strongly support the hypothesis that effective activation of human γδ T cells by nonpeptide Ags depends on the immunological synapse formation in which species-specific interactions take place. Nitrogen-containing bisphosphonate Ags are particularly intriguing, in that the vast majority of human tumor cells can efficiently present them to γδ T cells, resulting in the targeted lysis of tumor cells by γδ T cells. It is suggested that the putative molecules required for Ag presentation and effective γδ T cell activation are rather ubiquitously expressed on many human tumor cells. Molecular understanding of the interaction may provide a new clue for potential immunotherapy of human malignancy by targeting tumor cells with chemically defined nonpeptide Ags for γδ T cells.