We examined whether γδ T and αβ T cells accumulating in early B16 melanoma lesions regulate NK and NK T cells that attack tumor cells. Freshly isolated and cultured tumor-infiltrating lymphocyte (TIL) populations of NK and NK T cells lysed B16 and produced IFN-γ, whereas γδ T and a large part of αβ T cell populations had no substantial cytotoxicity against B16 and secreted Th2 cytokines. Furthermore, the freshly isolated NK1.1+ TIL population exhibited a higher anti-B16 effect than did splenocytes. γδ T and αβ T cell populations dramatically inhibited the cytotoxicity of NK and NK T cells in an MHC Kb-dependent manner. Culture supernatant from γδ T and αβ T cell populations inhibited the proliferation of NK and NK T cell populations but did not affect their cytotoxicity, suggesting that the released Th2 cytokines are merely partly involved in the down-modulation of NK-lineage cells. NK1.1+ cells obtained from TIL of γδ T cell-depleted mice significantly lysed B16 cells compared with those from control mice. Finally, anti-Kb Fab mAb injected intralesionally at an early, but not at a late, stage of development of B16 melanoma inhibited tumor growth. These findings suggest that Th2-type γδ T and αβ T cells infiltrating in early B16 development inhibit the tumoricidal activity of NK-lineage cells using their class I molecules and partly their suppressive cytokines.

It has been well known that several kinds of lymphocytes, e.g., NK, NK1.1+ αβTCR+ T (NK T), γδ T, and conventional αβ T cells, including Th cells and CTL, accumulate in tumor lesions at an early stage (1, 2). However, it remains obscure whether these cell populations affect each other in their functions because of difficulty in preparing a large number of fresh tumor-infiltrating lymphocytes (TILs)3 for analytical procedures. Th1 and Th2 cells and their released cytokines regulate each other as counterparts in several biologic events (3). Many observations have suggested that Th2 cytokines inhibit the process of Th1 cytokine-induced NK activation and CTL generation (4, 5, 6). There is much evidence that NK cells play a central role in early IFN-γ secretion in response to malignancies as well as infections (2, 7, 8). On the other hand, NK T cells, both CD4+ CD8 and CD4 CD8, provide the primary source of IL-4 by TCR engagement (9), although their NK1.1 stimulation leads to the production of a large amount of IFN-γ (10). γδ T cells can be classified into Th1 and Th2 types as well as αβ T cells (11). Therefore, it is possible that the tumoricidal activities of NK-lineage cells are inhibited by the Th2 cytokine-producing TILs in tumors sensitive to NK cells.

In addition to cytokines possibly elaborated from bystander lymphocytes, NK cell activity is profoundly influenced by MHC class I molecules on targets. It has been suggested that the differential sensitivity of tumor cells to NK cells may be inversely correlated to the expression of MHC class I molecules on some, but not all, target cells (12, 13). In fact, several NK cell-susceptible tumor cell lines, including YAC-1 lymphoma cells and B16 melanoma cells, acquire resistance to NK cytotoxicity upon transfection with class I genes or upon treatment with IFN-γ to augment class I expression (14, 15). In association with the class I expression, the susceptibility of tumor cells changes from NK cells to CTLs, since CTLs lyse tumor cells upon recognition of specific tumor peptide-class I complexes. In contrast, NK activity is often maximally inhibited in cytotoxicity assays using tumor cells expressing substantial levels of the autologous class I molecules (13, 16).

Murine inhibitory receptors with specificity for class I molecules expressed on NK and NK T cells (termed Ly49 families) have been identified as structurally homologous to C-type lectins (17, 18). Only one report has demonstrated that human Ig-like killer cell-inhibitory receptors are also expressed on murine NK cells (19). Mixed allogeneic lymphocyte studies revealed that NK cell subsets expressing Ly49C are functionally inhibited by MHC class I H-2Kb molecules derived from the H-2b allele (20). However, it remains unknown whether Ly49C can interact with the Db molecule. Ly49A is expressed on certain subsets of B6 mouse (H-2b) NK cells (21), but not on NK cells derived from H-2d or H-2k mice. Affinity-purified Kb and Db molecules, however, bind to Ly49A with low affinity (22), suggesting that relevant peptide-Kb or -Db complexes effectively bind to Ly49A and depress the function of NK cells (20, 22, 23). Thus, the maximal inhibition of NK and NK T cell cytotoxicities may be determined by the expression level of class I molecules or relevant peptide-class I complexes and/or Ly49 molecules.

B16 melanoma cells are a representative of NK-sensitive tumor cell lines, because they express very low levels of class I molecules (24, 25). Accordingly, B16 tumor cells more vigorously progress in NK cell-depleted mice (26). It has been demonstrated that NK1.1+ cells, including NK and NK T cells, vigorously accumulate early in the formation of B16 melanoma lesions (2). It is also noteworthy that B16 tumor cells do not regress despite the vigorous infiltration of NK and NK T cells at tumor sites. In the present study we demonstrate the mechanisms by which NK and NK T cells are functionally depressed at s.c. inoculated B16 tumor sites, where Kb expression is undetectable on the surface of B16 cells. Our results show that γδ T and αβ T cells that predominantly coinfiltrate in the tumor lesions inhibit the tumoricidal activities of NK and Th1-type NK T cells in a Kb-dependent manner and suppress the proliferation of these NK-lineage cells by releasing soluble factors, including Th2 cytokines.

Seven- to ten-week-old male C57BL/6 (B6) mice were obtained from Japan SLC (Hamamatsu, Japan), and maintained in our laboratory. B16 melanoma cells and YAC-1 lymphoma cells were maintained in DMEM (Nissui, Tokyo, Japan) supplemented with 10% FCS (Filtron, Brooklyn, Australia).

The following mAbs were purchased from PharMingen (San Diego, CA): FITC-conjugated anti-NK1.1 (PK136), anti-H-2Db (KH95), and anti-H-2Kb (AF6-88.5); phycoerythrin (PE)-conjugated anti-αβTCR (H57-597) and anti-γδTCR (GL3); purified form of anti-αβTCR (H57-597); anti-γδTCR (GL3); anti-H-2Kb (AF6-88.5); anti-H-2Db (KH95); anti-CD3 (145-2C11); anti-CD16/CD32 (2.4G2); rat IgG2b mAb (R35-38); anti-IFN-γ (R4-6A2); anti-IL-4 (BVD4-1D11) and anti-IL-10 (JES5-2A5) mAbs; and biotin-conjugated anti-IFN-γ (XMG1.2), anti-IL-4 (BVD6-24G2), and anti-IL-10 (SXC-1) mAbs. Affinity-purified mouse IgG and hamster IgG were obtained from Rockland (Gilbertsville, PA). The Fab form of anti-H-2Kb and anti-H-2Db mAbs were prepared using the ImmunoPure Fab preparation kit (Pierce, Rockford, IL) on the basis of papain cleavage of Igs. Hybridoma producing anti-γδTCR mAb (UC7-13D5; a gift from Dr. Bluestone, University of Chicago, Chicago, IL) and that producing anti-NK1.1 mAb (PK136) were cultured in DMEM supplemented with 10% FCS. UC7-13D5 and PK136 mAbs were purified from culture supernatant by ammonium sulfate precipitation and affinity chromatography with anti-hamster IgG-conjugated Sepharose or protein G-Sepharose, respectively.

RPMI 1640 (Nissui, Tokyo, Japan) medium supplemented with 25 mM HEPES, 2 mM l-glutamine, 1 mM nonessential amino acid, 5 × 10−2 mM 2-ME, 1 mM sodium pyruvate, 100 μg/l gentamicin (all from Life Technologies, Grand Island, NY), and 10% FCS (complete medium) was used in this study.

B16 melanoma cells (5 × 106) were inoculated s.c. into B6 mice. Tumors were resected on day 4, 5, 7, 14, or 20 after inoculation. TILs were prepared from B16 tumor suspensions by centrifugation with Histopaque 1083 (Sigma, St. Louis, MO). Ten milliliters of B16 tumor suspensions (1 × 105 cells/ml in PBS containing 10% FCS) applied on 5 ml of Histopaque 1083 were subjected to centrifugation at 1000 × g for 30 min at 20°C. The cells at the interface were collected, washed three times with DMEM, and used as B16 TILs. On flow cytometric analysis, 4-, 5-, 7-, 14- and 20-day TILs thus separated contained B16 cells at approximately 5, 5, 20, 30, and 30%, respectively.

Subpopulations of 5-day B16 TILs obtained from approximately 50 tumors (one tumor per mouse) were separated with immune magnetic beads. Anti-αβTCR (H57–597), anti-γδTCR (GL3), or anti-NK1.1 (PK136) mAb-conjugated magnetic beads were prepared by coupling tosil-activated immunomagnetic beads M-450 (Dynal, Chantilly, VA) with each mAb as described in the Dynal manual. After treatment with anti-CD16/32 Fc blocking mAb (2.4G2) at 37°C for 10 min, 5-day TILs (6 × 105) were mixed with anti-NK1.1 mAb-conjugated beads at a ratio of five beads per cell and incubated for 1 h at 37°C. The cells bound to magnetic beads were collected with a magnet. After three washings, the cells were separated from beads by 6-h cultivation with RPMI 1640 containing 10% FCS at 37°C in 5% CO2 and used as freshly isolated NK1.1+ cells (∼1.5 × 105, 62 and 36% populations of the cells were NK and NK T cells, respectively, as assessed by flow cytometry). αβ T and γδ T cell populations were further separated from the NK1.1+ cell-depleted 5-day TILs. The cells (4 × 105) were incubated with anti-αβTCR mAb-conjugated magnetic beads at a ratio of five beads per cell for 30 min, and then cells bound to beads were collected with a magnet. Unbound cells (2.5 × 105) were incubated with anti-γδTCR mAb-conjugated beads and collected under the same conditions. In both cases, the cells were separated from beads by 6-h cultivation as described above (αβ T cells, 1 × 105; γδ T cells, 1 × 105).

Cultured B16 TIL populations were prepared as follows. Five-day B16 TILs were cultured with complete medium supplemented with rIL-2 (20 U/ml; Genzyme, Boston, MA) for 5 days. During this cultivation, γδ T cells did not propagate, and NK and NK T cells expanded predominantly. These cultured TILs were mixed with anti-NK1.1 mAb-conjugated magnetic beads, and the bound cells were collected as described above. These NK1.1+ cells were further selected with anti-αβTCR mAb-conjugated beads and used as cultured NK T cells (NK1.1+ αβTCR+, 98% purity). The remaining cells were used as cultured NK cells (NK1.1+ αβTCR γδTCR, 84% purity). Cultured αβ T and γδ T cells were prepared from 5-day B16 TILs as follows. Anti-αβTCR mAb (H57-597)- or anti-γδTCR mAb (UC7-13D5)-immobilized plates were prepared by incubating each mAb (10 μg/well in 24-well plates) in 0.5 ml of 0.1 M carbonate buffer (pH 9.0) overnight at 4°C followed by washing three times with PBS. Freshly separated TILs were negatively selected with anti-NK1.1 mAb-conjugated magnetic beads. The remaining cells were cultured in complete medium on anti-αβTCR mAb- or anti-γδTCR mAb-coated plates (1 × 105 cells/well) for 5 days. The expanded cells were harvested and used as αβ T (NK1.1, αβTCR+ γδTCR; 99% purity) and γδ T (NK1.1, αβTCR γδTCR+; 89% purity) cells.

Splenic NK1.1+ cells were prepared by positively selecting with magnetic beads. Spleen cell suspensions from normal B6 mice were hemolyzed with 0.17 M ammonium chloride at 37°C for 5 min and incubated on a 5-cm plastic dish at 37°C for 90 min (1 × 106 cells/ml in complete medium) to remove dish-adherent cells. The nonadherent cells were positively selected with anti-NK1.1 mAb-conjugated magnetic beads at a ratio of three beads per cell and used as splenic NK1.1+ cells (NK1.1+ αβTCR, 83%; NK1.1+ αβTCR+, 10%).

Freshly purified and cultured TILs and subpopulations of TILs were stained with FITC- and/or PE-conjugated mAbs for 30 min at 4°C. After washing three times, the TILs were analyzed with a flow cytometer (FACScan, Becton Dickinson, Oxnard, CA). All procedures were conducted after blocking the nonspecific binding with anti-CD16/CD32 mAb. The mononuclear cell fraction was gated to exclude contaminating B16 tumor cells, and data were displayed on two-color contour plots or histogram by FACScan programs. To analyze H-2 expression of freshly purified B16 tumor cells, B16 tumor suspensions were centrifuged with Histopaque 1083 (1000 × g, 30 min), and pelleted cells were collected and stained with FITC-conjugated anti-Kb, anti-Db, or control mouse IgG mAb for 30 min at 4°C following anti-CD16/32 mAb treatment. The fluorescence intensity was visualized on histogram by flow cytometric analysis.

The cytokine profiles of NK, NK T, αβ T, and γδ T cell populations in B16 TILs were examined by ELISPOT assay as described previously (27). One microgram per milliliter in 100 μl of 0.1 M carbonate buffer (pH 9.0) of anti-IFN-γ (R4-6A2), anti-IL-4 (BVD4-1D11), or anti-IL-10 (JES5-2A5) mAb was added to each well of 96-well ELISPOT plates (MultiScreen-HA, Millipore, Bedford, MA) and incubated at 4°C for 12 h. After coating, plates were washed twice with PBS, blocked with PBS containing 10% FCS at 37°C for 1 h, and washed twice with PBS. Freshly isolated B16 TILs and their separated populations were cultured for 24 h in RPMI 1640 medium supplemented with 1 μg/ml Con A. The cells (5 × 103) were further cultured overnight in Ab-coated ELISPOT plates at 37°C in 5% CO2. Plates were then vigorously washed 10 times with PBS and incubated with 0.5 μg/ml in 100 ml of PBS containing 10% FCS of biotin-conjugated mAb (anti-IFN-γ (XMG1.3), anti-IL-4 (BVD6-24G2), or anti-IL-10 (SXC-1) mAb) at 37°C for 2 h. Following five washes with PBS, the plates were incubated with streptavidin-peroxidase (Boehringer Mannheim, Mannheim, Germany; 1/1000 in PBS containing 10% FCS) at 37°C for 1 h. After washing five times with PBS, 100 μl of substrate (1 mg/ml of 3,3′-diaminobenzidine tetrahydrochloride containing 0.003% H2O2; Sigma) was added to each well and incubated at 37°C for 15 min. Developed spots were counted using a dissecting microscope.

To test class I-dependent inhibition of NK cells at tumor sites, 100 μg (in 50 μl of PBS)/mouse of anti-H-2Kb Fab mAb, anti-H-2Db Fab mAb, or mouse IgG Fab Ab was injected intralesionally at tumor sites on 3 consecutive days, i.e., on days 4 to 6 or days 12 to 14 after s.c. inoculation of B16 cells.

To obtain γδ T cell-depleted B6 mice, 500 μg/mouse of anti-γδTCR (UC7-13D5) or hamster IgG Ab as a control was administered i.v. to B6 mice. The disappearance of γδTCR+ cells in the Ab-treated mice was confirmed by flow cytometric analysis of splenocytes and PBMC compared with those from intact or hamster IgG-treated mice.

NK1.1+ cell-depleted B6 mice were prepared as described previously (2). Mice were injected i.v. with 100 μg of anti-NK1.1 (PK136) mAb. Five days after Ab treatment, 5 × 106 B16 cells were s.c. inoculated. The depletion of NK1.1+ cells, including NK and NK T cells, was confirmed by flow cytometric analysis of 5-day TILs compared with those from intact or mouse IgG2a-treated mice.

Varying numbers of separated TIL populations, NK, NK T, αβ T, or γδ T cells, were assayed by incubation with 1 × 104 51Cr-labeled freshly isolated or cultured B16 or YAC-1 for 5 h at 37°C. Otherwise, the cytotoxicities of separated αβ T and γδ T cells were examined by incubation with 1 × 104 51Cr-labeled cultured NK or NK T cells for 5 h at 37°C. Target cells were radiolabeled by suspension at a concentration of 1 × 106 cells/ml in medium containing 200 μCi/ml Na51Cr (DuPont-New England Nuclear, Boston, MA) for 60 min at 37°C and were washed three times. After the incubation, the radioactivity in the medium and cells was counted in a gamma counter. The percent lysis was calculated as described previously (28). In all cytotoxicity assay, spontaneous 51Cr release values were approximately 8% (B16), 4% (YAC-1), and 11% (NK, NK T cells) of the maximal release after 5-h cultivation.

In separate experiments, the modulatory effects of γδ T or αβ T cells on NK and NK T cell cytotoxicities against B16 and YAC-1 were tested by the addition of freshly isolated or cultured γδ T or αβ T cells to the cytotoxicity assay in varying numbers.

In some experiments, cultured or freshly isolated γδ T or αβ T cells were cultured in 96-well plates at 37°C for 1 day (5 × 104/well in complete medium) to obtain culture supernatants, which were added to the cytotoxicity assay of NK and NK T cells against B16.

The cultured NK and NK T cells (2 × 105 cells/well) were incubated in triplicate for 24 h in 96-well plates (Corning, Corning, NY) in 100 μl of complete medium. [3H]TdR (Amersham, Arlington Heights, IL; 1 μCi/well) was added to the culture 8 h before harvest. The cells were harvested on glass-fiber filters using a cell harvester (Cambridge Technologies, Watertown, MA), and their radiouptake was measured in a scintillation counter. Culture supernatants from γδ T or αβ T cells prepared as described above were added to the NK or NK T cell proliferation assay in varying volumes.

To determine class I-dependent αβ T or γδ T cell inhibition of NK or NK T cell cytotoxicity, αβ T or γδ T cells were preincubated with Kb- and Db-specific Fab mAb or control mouse IgG Fab Ab (10 μg/ml). After washing three times with DMEM, the Ab-treated αβ T or γδ T cells were added to NK and NK T cell cytotoxicity assays in varying numbers. In separate experiments, B16, NK, and NK T cells were treated with Kb- and Db-specific Fab mAb or control mouse IgG Fab Ab (10 μg/ml) and then washed three times. These Ab-treated cells were used as effector or target cells in the NK or NK T cell cytotoxicity assay.

TILs were separated from B16 tumor suspensions by centrifugation with Histopaque at various time points after s.c. inoculation of B16, and they were phenotyped by flow cytometry. As shown in Figure 1,A, NK (NK1.1+, αβTCR), NK T (NK1.1+, αβTCR+), αβ T (NK1.1, αβTCR+), and γδ T cells (NK1.1, γδTCR+) were present at 32, 21, 11, and 17%, respectively, of the total 5-day TILs. A small number of NK1.1+ γδTCR+ cells also infiltrated (0.8%). Furthermore, the percentages of these four major populations peaked on days 5 to 7 after tumor cell inoculation and gradually decreased thereafter (Fig. 1 B). Therefore, NK, NK T, αβ T, and γδ T cells transiently accumulated soon after tumor cell implantation.

FIGURE 1.

Accumulation of NK, NK T, αβ T, and γδ T cells at the early stage of B16 development. A, Freshly isolated 5-day B16 TILs were double stained with PE-conjugated anti-αβTCR or anti-γδTCR mAb and FITC-conjugated anti-NK1.1 mAb and analyzed by flow cytometry. Numbers represent the percentage of total cells in each quadrant. B, B16 TILs were separated on varying days after inoculation and stained with FITC-conjugated anti-NK1.1 mAb or PE-conjugated anti-αβTCR or anti-γδTCR mAb, and their numbers were counted by flow cytometry. Total TILs were enumerated by gating mononuclear cell population. Data represent percentages of TILs, NK1.1+, αβTCR+, and γδTCR+ cells in the B16 tumor suspension. PE-conjugated mouse IgG (□) and hamster IgG (▵) were used as controls.

FIGURE 1.

Accumulation of NK, NK T, αβ T, and γδ T cells at the early stage of B16 development. A, Freshly isolated 5-day B16 TILs were double stained with PE-conjugated anti-αβTCR or anti-γδTCR mAb and FITC-conjugated anti-NK1.1 mAb and analyzed by flow cytometry. Numbers represent the percentage of total cells in each quadrant. B, B16 TILs were separated on varying days after inoculation and stained with FITC-conjugated anti-NK1.1 mAb or PE-conjugated anti-αβTCR or anti-γδTCR mAb, and their numbers were counted by flow cytometry. Total TILs were enumerated by gating mononuclear cell population. Data represent percentages of TILs, NK1.1+, αβTCR+, and γδTCR+ cells in the B16 tumor suspension. PE-conjugated mouse IgG (□) and hamster IgG (▵) were used as controls.

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We examined whether each of four 5-day TIL populations has the ability to lyse B16 cells. Short term cultured (C-) NK, NK T, αβ T, and γδ T populations or freshly isolated (F-) NK1.1+, αβ T, and γδ T populations were prepared from 5-day TILs as described in Materials and Methods. A 5-h cytotoxicity assay of these populations was performed against fresh B16 isolated from tumor lesions (F-B16), cultured B16 (C-B16), or YAC-1. C-NK and F-NK1.1+ cells vigorously lysed F-B16, C-B16, and YAC-1 (Fig. 2 A). The cytolytic profile of C-NK T cells was similar, but at weak levels, to that of NK cells, although they did not exhibit an anti-C-B16 effect. In contrast, C- and F-αβ T and C- and F-γδ T cells had no cytotoxicity against any tumor target.

FIGURE 2.

Cytotoxicity of TILs and NK, NK T, αβ T, and γδ T cell populations. A, Short term 5-day C-B16 tumor-infiltrating NK, NK T, αβ T, and γδ T cell populations (effector) were assayed for 5 h with 51Cr-labeled F-B16, cultured C-B16 cells, or YAC-1 cells (target) at the indicated E:T cell ratios. Freshly isolated NK1.1+, αβ T, and γδ T cell populations of 5-day B16 TILs (effector) were also assayed for 5 h with 51Cr-labeled F-B16 cells (target) at an E:T cell ratio of 5. B, Freshly isolated 5-day B16 TILs, their NK1.1+ cell population and the NK1.1+ cell-removed remainder, and the splenic NK1.1+ cell population of normal B6 mice were assayed with 51Cr-labeled F-B16 cells at an E:T cell ratio of 5. The figure shows one experiment of three performed with similar results. Data are expressed as the mean ± SE of duplicate experiments.

FIGURE 2.

Cytotoxicity of TILs and NK, NK T, αβ T, and γδ T cell populations. A, Short term 5-day C-B16 tumor-infiltrating NK, NK T, αβ T, and γδ T cell populations (effector) were assayed for 5 h with 51Cr-labeled F-B16, cultured C-B16 cells, or YAC-1 cells (target) at the indicated E:T cell ratios. Freshly isolated NK1.1+, αβ T, and γδ T cell populations of 5-day B16 TILs (effector) were also assayed for 5 h with 51Cr-labeled F-B16 cells (target) at an E:T cell ratio of 5. B, Freshly isolated 5-day B16 TILs, their NK1.1+ cell population and the NK1.1+ cell-removed remainder, and the splenic NK1.1+ cell population of normal B6 mice were assayed with 51Cr-labeled F-B16 cells at an E:T cell ratio of 5. The figure shows one experiment of three performed with similar results. Data are expressed as the mean ± SE of duplicate experiments.

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The anti-B16 cytotoxicity of fresh 5-day B16 TILs were further investigated in a comparison between F-NK1.1+ 5-day TILs and F-NK1.1+ normal splenocytes. The F-TIL exhibited a weak cytotoxicity against F-B16. The NK1.1+ cell separated from TILs killed F-B16 more efficiently than the splenic NK1.1+ population (Fig. 2 B). In addition, F-TILs depleted of NK1.1+ cells did not exhibit significant cytotoxicity.

Taken together, these results indicated 1) that NK cells and NK T cells are the major cytotoxic effectors against B16 at the early stage of tumor development, with the former being more effective than the latter; 2) that NK cells are in an activated state in the B16 tumor lesion; and 3) that NK cell cytotoxicity is potentially inhibited by some TIL populations coinfiltrating with NK cells.

By an in vitro study using each of the four populations of short term cultured 5-day TILs, we determined whether NK and NK T cell cytotoxicities are abolished by the addition of αβ T or γδ T cells. NK and NK T cell cytotoxicities against C-B16 and YAC-1, respectively, were dose dependently decreased by the αβ T cell or γδ T cell population, although the inhibitory effect of γδ T cells was stronger than that of αβ T cells (Fig. 3,A). The NK cell cytotoxicity was absolutely abrogated at a γδ T cell/NK cell ratio of 1 (5 × 104/well). This inhibition was not due to direct killing because neither γδ T nor αβ T cell populations lysed NK or NK T cells as assessed by cytotoxic assay against 51Cr-labeled NK or NK T cells (Fig. 3,B). The proliferation of NK and NK T cell populations was quickly decreased after coculture with the αβ T or γδ T cell population (Fig. 3 C). Again, the suppressive ability of γδ T cells was higher than that of αβ T cells.

FIGURE 3.

Inhibitory effects of 5-day B16 tumor-infiltrating αβ T and γδ T cells on the cytotoxicity and proliferation of NK and NK T cells. A, The short term cultured αβ T or γδ T cell population of 5-day B16 TILs was added to the NK cytotoxicity assay against B16 or to the NK T cytotoxicity assay against YAC-1 cells in the indicated numbers. Data are representative results for three independent experiments. B, Short term cultured αβ T and γδ T cell populations of 5-day B16 TILs were assayed with a 51Cr-labeled cultured NK or NK T cell population of 5-day B16 TILs for 5 h at the indicated ratios. Data are expressed as the mean ± SE of two independent experiments. C, Short term cultured NK and NK T cells of 5-day B16 TILs (2 × 105 cells/well) were cocultured with the indicated numbers of mitomycin C-treated αβ T or γδ T cells for 24 h, and then [3H]thymidine incorporation was measured by 8-h incubation. Data are expressed as the mean ± SD of triplicate cultures.

FIGURE 3.

Inhibitory effects of 5-day B16 tumor-infiltrating αβ T and γδ T cells on the cytotoxicity and proliferation of NK and NK T cells. A, The short term cultured αβ T or γδ T cell population of 5-day B16 TILs was added to the NK cytotoxicity assay against B16 or to the NK T cytotoxicity assay against YAC-1 cells in the indicated numbers. Data are representative results for three independent experiments. B, Short term cultured αβ T and γδ T cell populations of 5-day B16 TILs were assayed with a 51Cr-labeled cultured NK or NK T cell population of 5-day B16 TILs for 5 h at the indicated ratios. Data are expressed as the mean ± SE of two independent experiments. C, Short term cultured NK and NK T cells of 5-day B16 TILs (2 × 105 cells/well) were cocultured with the indicated numbers of mitomycin C-treated αβ T or γδ T cells for 24 h, and then [3H]thymidine incorporation was measured by 8-h incubation. Data are expressed as the mean ± SD of triplicate cultures.

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B16 cells were inoculated s.c. in γδ T cell-depleted mice prepared by i.v. administration of anti-γδTCR mAb (UC7–13D5; 500 μg/mouse). The cytotoxicity of 5-day F-TILs (NK1.1+ αβTCR, 56%; NK1.1+ αβTCR+, 22%; NK1.1 αβTCR+, 9%) from these mice was assayed against that of B16. The TILs from γδ T cell-depleted mice lysed significant numbers of F-B16, C-B16, or YAC-1 tumor cells compared with those from hamster IgG-treated mice (Fig. 4). When NK1.1+ cells were removed, these TILs failed to lyse B16. These findings suggested that B16 cells escape from NK and NK T cell attack by virtue of γδ T cells and partly by αβ T cells in tumor lesions.

FIGURE 4.

Enhanced anti-B16 cytotoxicity of 5-day B16 TILs freshly isolated from γδ T cell-depleted mice. Fresh 5-day B16 TILs were separated from γδ T-depleted or control hamster IgG-treated mice. NK1.1+ cell-removed fractions of TILs from γδ T cell-depleted mice were prepared by negative selection with anti-NK1.1 mAb-conjugated magnetic beads. These cells were assayed using 51Cr-labeled F- or C-B16 or YAC-1 at the indicated ratios. The figure shows one experiment of three performed with similar results. Data are expressed as the mean ± SE of results of duplicate experiments.

FIGURE 4.

Enhanced anti-B16 cytotoxicity of 5-day B16 TILs freshly isolated from γδ T cell-depleted mice. Fresh 5-day B16 TILs were separated from γδ T-depleted or control hamster IgG-treated mice. NK1.1+ cell-removed fractions of TILs from γδ T cell-depleted mice were prepared by negative selection with anti-NK1.1 mAb-conjugated magnetic beads. These cells were assayed using 51Cr-labeled F- or C-B16 or YAC-1 at the indicated ratios. The figure shows one experiment of three performed with similar results. Data are expressed as the mean ± SE of results of duplicate experiments.

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Fresh NK1.1+, αβ T, and γδ T cells separated from 5-day B16 TILs were stimulated with Con A (1 μg/ml), and the cytokine profiles of each population was investigated by ELISPOT assay. NK1.1+ cells including NK and NK T cell populations markedly expressed IFN-γ, and only a few of them (3.7% of the total number) had the ability to produce IL-4 and IL-10 (Fig. 5,A). On the contrary, αβ T and γδ T cell populations secreted IL-4 and IL-10 at high levels, with 8.9% of the total αβ T cell number weakly expressing IFN-γ. Furthermore, short term cultivation of each population did not influence on its cytokine profile (NK: IL-4, 0%; IFN-γ, 88%; NK T: IL-4, 2.8%; IFN-γ, 72%; αβ T: IL-4, 65%; IFN-γ, 14%; γδ T: IL-4, 83%; IFN-γ, 0%). ELISPOT assay was also performed on 5-day F-B16 TILs. A large number of F-B16 TILs (57%) produced IL-4 and IL-10, whereas the number of IFN-γ-producing cells was small (3.2%; Fig. 5,B). This raised the possibility that Th2 cytokine-producing αβ T and γδ T cells inhibit Th1-type NK and NK T cell activities by releasing cytokines. Therefore, the modulatory effects of γδ T and αβ T cell culture supernatants on cytotoxicity and proliferation of NK and NK T cells were tested. The results are shown in Figure 6. Although the proliferation of NK and NK T cells was decreased by γδ T or αβ T cell culture supernatant, the supernatants exerted no inhibitory effect on NK and NK T cell cytotoxicity. Thus, it is likely that cytokines produced by γδ T and αβ T cells are only partly involved in the mechanisms by which they inhibit NK and NK T cell cytotoxicity.

FIGURE 5.

Cytokine profile of each T cell population of 5-day B16 TILs or 5-day F-B16 TILs. A, The freshly isolated NK1.1+, αβ T or γδ T cell population of 5-day B16 TILs was stimulated overnight with Con A (1 μg/ml), and then IL-4-, IL-10-, or IFN-γ-producing cells (5 × 103 in duplicate) were enumerated by ELISPOT assay. B, Five-day F-B16 TILs (5 × 103 in duplicate) were assayed as described above. In both experiments, visualized spots were counted under a microscope, and the percentage of cytokine-producing cells was calculated. Data are expressed as the mean ± SE of results of duplicate experiments.

FIGURE 5.

Cytokine profile of each T cell population of 5-day B16 TILs or 5-day F-B16 TILs. A, The freshly isolated NK1.1+, αβ T or γδ T cell population of 5-day B16 TILs was stimulated overnight with Con A (1 μg/ml), and then IL-4-, IL-10-, or IFN-γ-producing cells (5 × 103 in duplicate) were enumerated by ELISPOT assay. B, Five-day F-B16 TILs (5 × 103 in duplicate) were assayed as described above. In both experiments, visualized spots were counted under a microscope, and the percentage of cytokine-producing cells was calculated. Data are expressed as the mean ± SE of results of duplicate experiments.

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FIGURE 6.

Inhibition of the proliferation, but not the cytotoxicity, of NK and NK T cell populations by culture supernatants from γδ T and αβ T cell populations. Culture supernatants of short term cultured γδ T or αβ T cell population of 5-day B16 TILs were added to the NK or NK T cell proliferation assay and the cytotoxicity assay against B16 at the indicated final concentration. Data are expressed as the mean ± SE of results from experiments performed in duplicate.

FIGURE 6.

Inhibition of the proliferation, but not the cytotoxicity, of NK and NK T cell populations by culture supernatants from γδ T and αβ T cell populations. Culture supernatants of short term cultured γδ T or αβ T cell population of 5-day B16 TILs were added to the NK or NK T cell proliferation assay and the cytotoxicity assay against B16 at the indicated final concentration. Data are expressed as the mean ± SE of results from experiments performed in duplicate.

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We examined the possibility that inhibition of NK and NK T cell cytotoxicities is mediated by MHC class I molecules on the αβ T and γδ T cells. The 5-day C-αβ T or C-γδ T cell population preincubated with anti-Kb Fab mAb was added to the C-NK or C-NK T cell cytotoxicity assay against F-B16 in varying numbers. As shown in Figure 7,A, Kb-masked αβ T and γδ T cells did not inhibit either NK or NK T cell cytotoxicity at any αβ T or γδ T cell number (1, 2.5, and 5 × 104 cells). In contrast, Db-masked or control mouse IgG Fab mAb-treated αβ T and γδ T cells still retained their inhibitory activities for both NK and NK T cell cytotoxicities at levels comparable to those of untreated αβ T or γδ T cells. Furthermore, splenic γδ T cells upon stimulation with anti-γδTCR mAb (UC7-13D5) also inhibited NK and NK T cell cytotoxicities in a Kb-dependent manner; freshly isolated splenic γδ T cells showed no inhibition (Fig. 7 B), suggesting that these NK and NK T cells can be inhibited by Kb molecules only when γδ T and αβ T cells are activated. These results indicate that αβ T or γδ T cells inhibit NK and NK T cell cytotoxicities in a Kb molecule-dependent fashion.

FIGURE 7.

Inhibition of NK and NK T cell cytotoxicity by αβ T and γδ T cells in a Kb-dependent fashion. A, An anti-Kb- or anti-Db-treated or control mouse IgG-treated, cultured γδ T or αβ T cell population of 5-day B16 TILs was added to NK or NK T cell cytotoxicity assays against F-B16 at the indicated number. NK and NK T cytotoxicity assays were performed at an E:T cell ratio of 5. B, Untreated or Kb-, Db-, or control mouse IgG-treated freshly isolated or stimulated splenic γδ T cell population was added to NK cytotoxicity assay against B16 (E:T cell ratio of 5) at the indicated number. Data are expressed as the mean ± SE of results from experiments performed in duplicate. C, B16 tumor cells freshly prepared from 5-day B16 tumors were stained with FITC-conjugated anti-Kb (solid line), Db (dashed line), or control mouse IgG (dotted line) mAb, and analyzed by flow cytometry. D, Fresh B16, cultured NK, or cultured NK T cells of 5-day B16 TILs were treated with anti-Kb Fab mAb. These Kb-masked cells were used as target or effector cells for NK or NK T cytotoxicity assay against fresh B16. Data are expressed as the mean ± SE of results from experiments performed in duplicate.

FIGURE 7.

Inhibition of NK and NK T cell cytotoxicity by αβ T and γδ T cells in a Kb-dependent fashion. A, An anti-Kb- or anti-Db-treated or control mouse IgG-treated, cultured γδ T or αβ T cell population of 5-day B16 TILs was added to NK or NK T cell cytotoxicity assays against F-B16 at the indicated number. NK and NK T cytotoxicity assays were performed at an E:T cell ratio of 5. B, Untreated or Kb-, Db-, or control mouse IgG-treated freshly isolated or stimulated splenic γδ T cell population was added to NK cytotoxicity assay against B16 (E:T cell ratio of 5) at the indicated number. Data are expressed as the mean ± SE of results from experiments performed in duplicate. C, B16 tumor cells freshly prepared from 5-day B16 tumors were stained with FITC-conjugated anti-Kb (solid line), Db (dashed line), or control mouse IgG (dotted line) mAb, and analyzed by flow cytometry. D, Fresh B16, cultured NK, or cultured NK T cells of 5-day B16 TILs were treated with anti-Kb Fab mAb. These Kb-masked cells were used as target or effector cells for NK or NK T cytotoxicity assay against fresh B16. Data are expressed as the mean ± SE of results from experiments performed in duplicate.

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There remained the possibility that Kb molecules expressed on B16, and NK and NK T cells participate in the inhibition of NK and NK T cell cytotoxicities. By flow cytometric analysis, F-B16 cells did not express a detectable level of Kb molecules, although they had a low level of Db molecules (Fig. 7,C). Moreover, Kb-masking of F-B16 with specific mAb did not affect the cytolytic efficacy of C-NK or C-NK T cells (Fig. 7 D). Likewise, the cytotoxic activities of NK and NK T cells were unchanged after Kb-specific Fab mAb treatment. Therefore, the results excluded the above possibility.

On the basis of the concept that γδ T and αβ T cells exert an anti-NK and NK T cell action with their Kb molecules, we had anticipated that administration of Kb-specific Fab mAb into a tumor restores NK and NK T cell cytotoxicities, resulting in inhibition of tumor growth. NK-depleted mice were obtained by i.p. administration of anti-NK1.1 mAb (100 μg/mouse) as previously described (2). Kb- or Db-specific Fab mAb was injected intralesionally into s.c. inoculated B16 melanoma lesions of untreated or NK-depleted B6 mice. In untreated B6 mice, B16 growth was significantly inhibited by Kb-specific mAb that was injected consecutively on days 4 to 6 after tumor inoculation, whereas the growth was not affected by Db-specific or control mAb (Fig. 8). However, administration of anti-Kb mAb on days 12 to 14 failed to inhibit B16 growth (data not shown). This finding is in accordance with the observation that γδ T and αβ T cells accumulated in the B16 lesion markedly 5 to 7 days after tumor inoculation (see Fig. 1,B). In contrast, Kb-dependent inhibition of B16 growth was not found in the NK-depleted mice (Fig. 8). These in vivo results further suggest a critical role for Kb molecules on γδ T and αβ T cells in the inhibition of NK and NK T cells.

FIGURE 8.

Inhibition of B16 growth by administration of Kb-specific Fab mAb into melanoma lesions composed of Kb-nonexpressing B16 cells. Anti-Kb, anti-Db, or mouse IgG Fab mAb was injected into B16 melanoma lesions in NK1.1+ cell-depleted or untreated B6 mice on days 4, 5, and 6 after s.c. tumor inoculation. Subsequent tumor growth was measured. Vertical bars represent the SEM for five mice in each group. Data are representative results from three independent experiments.

FIGURE 8.

Inhibition of B16 growth by administration of Kb-specific Fab mAb into melanoma lesions composed of Kb-nonexpressing B16 cells. Anti-Kb, anti-Db, or mouse IgG Fab mAb was injected into B16 melanoma lesions in NK1.1+ cell-depleted or untreated B6 mice on days 4, 5, and 6 after s.c. tumor inoculation. Subsequent tumor growth was measured. Vertical bars represent the SEM for five mice in each group. Data are representative results from three independent experiments.

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The present study demonstrates how B16 cells evade the attack of NK and NK T cells that infiltrate in the tumor at an early stage. Among TILs, NK and NK T cells had a cytotoxic effect on B16 cells, and γδ T and αβ T cells coinfiltrating with these cytotoxic cells inhibited their anti-B16 activities and thus appeared to promote tumor growth. Two mechanisms by which γδ T and αβ T cells inhibit the NK-lineage cells are proposed from our study. First, since the former two T cell populations belonged to Th2 cells and NK and NK T cells were of the Th1 type, Th2 cytokines produced by γδ T and αβ T cells may mediate the inhibition. Second, more importantly, Kb molecules expressed on these bystander T cells mediate dysfunctioning of NK and NK T cell cytotoxicites.

It has been shown that NK cells is a major IFN-γ source in innate immune reactions (2, 7, 8). Numerous studies have revealed an inhibitory effect of IL-4 on the activation process of NK cells mediated by IL-2, IL-12, and IL-15 (4, 5, 29). On the other hand, it remains obscure whether Th1-type NK T cells, secreting only IFN-γ upon NK1.1 stimulation (10), are suppressed by Th2 cytokines or another Th2-type NK T cell population, secreting a large amount of IL-4 upon TCR engagement (9). This study clearly demonstrated that Th1-type NK T cells as well as NK cells infiltrate in the early B16 lesion, and their proliferations were also inhibited by Th2-type γδ T and αβ T cells. However, this does not seem to be critical for the evasion mechanisms of B16 from NK1.1+ cell attack; culture supernatants from these T cell populations, although inhibitory for the proliferation of the NK-lineage cells, did not depress their cytotoxicities, suggesting that Th2 cytokines participate only partly in the inhibition.

B16 tumor cells could not directly attenuate NK cell activity via their class I molecules because of lack of Kb expression. Instead, γδ T and αβ T cells that accumulated in B16 tumor lesions down-modulated the cytotoxicity of NK cells. This down-regulation was restricted by Kb molecules expressed on these coinfiltrating T cells. In addition, our study demonstrated that activated, but not resting, splenic γδ T cells from naive mice inhibited NK cell activity in a Kb-dependent manner. One possible explanation for these findings is that some endogenous peptides synthesized in activated γδ T and αβ T cells can inhibit NK and NK T cell activities when presented in the context of the Kb molecule. There have been a number of reports concerning the peptide-dependent NK cell inhibition in human systems (30, 31, 32). In murine models, participation of peptides in NK cell inhibition is also suggested, as Kb gene-transfected NK-sensitive tumor cells that conjunctively express peptides acquire resistance against NK cell attack (14, 15). Although, the binding affinity of purified Kb molecule to Ly49 is too low to negatively regulate Ly49+ NK cells (22), Kb-restricted peptides induced in activated γδ T and αβ T cells are possibly able to alter the Kb structure so that it binds with high affinity to NK cell inhibitory receptors such as Ly49. These peptides should be identified to fully understand how NK- and NK T-sensitive tumor cells evade immunosurveillance at the molecular level. Alternatively. in cooperation with Kb molecules on γδ T and αβ T cells, some adhesion molecules, whose expression is potentially elevated when these lymphocytes are activated (33), may give rise to strong binding to NK and NK T cells, leading to maximal inhibition of their cytotoxicities. In this case, it is also speculated that Ly49 recognizes Kb molecules without endogenous peptides.

It remains to be elucidated whether the γδ T and αβ T cells accumulating in early B16 tumor lesions recognize B16 cells. γδTCRs can recognize not only classical MHC class I complexes (34) in the same way as target recognition by αβTCR of CTLs, but also highly conserved protein family such as heat shock proteins (35) and nonclassical class I molecules, including CD1, Qa-1, and TL (36, 37, 38). These substances are produced by cells exposed to stressful stimuli, heat, starvation, infection, and malignant transformation (39). Thus, it is possible that the B16 tumor-infiltrating γδ T cells interact with these molecules expressed on the B16 tumor cells.

NK1.1 molecule is a receptor for oligosaccharide on target cells, and its ligation activates NK-lineage cells (40, 41). Accordingly, B16 lysis by NK cells is blocked by the addition of Fab mAb for NK1.1 (42). Our study showed that only the NK cell population, not the NK T cell population, lysed cultured B16 cells in the 5-h cytotoxicity assay, although both populations expressed comparable levels of NK1.1 molecules. Freshly isolated B16 cells, however, were cytolysed even by NK T cells. Another study has revealed that fresh B16 cells express Fas molecules more dramatically than those on cultured B16 (N. Seo et al., unpublished observation). While several reports have shown that NK and NK T cells lyse tumor targets both with cytotoxic granules, such as perforin and granzyme B, and through a Fas-dependent pathway (43), some NK T cell populations, but not all, seem to predominantly use the Fas-mediated pathway (44, 45). Therefore, it is supposed that B16 cells are more effectively lysed by NK T cells in a Fas-dependent manner (45), although both NK and NK T cells can probably recognize B16 cells via NK1.1 molecules. Nevertheless, since NK T cells with αβTCR can recognize CD1 molecules that are broadly found in lymphoid and nonlymphoid tissues (46, 47), the mechanism of B16 cell recognition by NK T cells seems to be more complex than that by NK cells. Thus, the mechanism of target recognition of NK T cells is now controversial (48). However, NK T cells are assumed to recognize B16 cells via only NK1.1 molecules, but not via αβTCR, in tumor lesions, because, as indicated in this study, the NK T cell population isolated from B16 TILs secreted IFN-γ, but not IL-4 or IL-10, as shown in a cytokine production pattern induced by NK1.1 ligation (10).

Immunoregulatory roles for γδ T cells have been well studied in various systems (28, 49, 50, 51). γδ T cells seem to act as suppressors of cytotoxic cells in tumor environments. γδ T cells purified from tumor-bearing mice inhibit CTL activities through an unknown factor(s) (28). In addition, our study provides evidence that γδ T cells, via their Kb molecules, block the cytotoxicity of NK and NK T cells in the B16-bearing animal model. Of interest is the observation that these γδ T cells have a weak cytotoxicity against tumor cells, in contrast to their cytotoxic effector roles against malignancies as described previously (52, 53, 54, 55). Thus, γδ T cells seem to be functionally classifiable as cytotoxic cells and immunomodulatory cells, in particular inhibitory cells in tumor-bearing animals. As indicated in our report and another study (11), Th1- and Th2-type γδ T cells may be classified into the cytotoxic effector and the immunoregulator, respectively.

We thank Ms. Keiko Sugaya for technical assistance and Ms. Fumiyo Ohmori for preparation of the manuscript.

1

This work was supported in part by a grant from the Ministry of Health of Japan and by the Shiseido fund.

3

Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; PE, phycoerythrin; ELISPOT, enzyme-linked immunospot; C-, cultured; F-, freshly isolated.

1
Kowalczyk, D., W. Skorupski, Z. Kwias, J. Nowak.
1996
. Activated γ/δ T lymphocytes infiltrating renal cell carcinoma.
Immunol. Lett.
53
:
15
2
Tamada, K., M. Harada, K. Abe, T. Li, H. Tada, Y. Onoe, K. Nomoto.
1997
. Immunosuppressive activity of cloned natural killer (NK1.1+) T cells established from murine tumor-infiltrating lymphocytes.
J. Immunol.
158
:
4846
3
Mosmann, T. R., R. L. Coffman.
1989
. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties.
Annu. Rev. Immunol.
7
:
145
4
Nagler, A., L. L. Lanier, J. H. Phillips.
1988
. The effects of IL-4 on human natural killer cells: a potent regulator of IL-2 activation and proliferation.
J. Immunol.
141
:
2349
5
Spits, H., H. Yssel, X. Paliard, R. Kastelein, C. Figdor, J. E. De Vries.
1988
. IL-4 inhibits IL-2-mediated induction of human lymphocyte-activated killer cells, but not the generation of antigen-specific cytotoxic T lymphocytes in mixed leukocyte culture.
J. Immunol.
141
:
29
6
Kelsall, B. L., E. Stuber, M. Neurath, W. Strober.
1996
. Interleukin-12 production by dendritic cells: the role of CD40-CD40L interactions in Th1 T-cell responses.
Ann. NY Acad. Sci.
795
:
116
7
Scharton, T. M., P. Scott.
1993
. Natural killer cells are a source of interferon-γ that derives differentiation of CD4+ T cell subsets and induces early resistance to Leishmania major in mice.
J. Exp. Med.
178
:
567
8
Trinchieri, G..
1994
. Recognition of major histocompatibility complex class I antigens by natural killer cells.
J. Exp. Med.
180
:
417
9
Yoshimoto, T., W. E. Paul.
1994
. CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3.
J. Exp. Med.
179
:
1285
10
Arase, H., N. Arase, T. Saito.
1996
. Interferon-γ production by natural killer cells and NK1.1+ T cells upon NKR-P1 cross-linking.
J. Exp. Med.
183
:
2391
11
Wen, L., D. F. Barber, W. Pao, F. S. Wong, M. J. Owen, A. Hayday.
1998
. Primary γδ cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation.
J. Immunol.
160
:
1965
12
Bjorkman, P. J., M. A. Saper, B. Samuraoui, W. S. Bennet, J. L. Strominger, D. C. Wiley.
1987
. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens.
Nature
329
:
512
13
Kesari, V. K., J. Geliebter.
1993
. Rejuvenated expression of H-2Kb in RMA-S cells does not affect alloreactive T cell- and natural killer cell-mediated lysis.
Immunol. Lett.
38
:
77
14
Carlow, D. A., U. Payne, N. Hozumi, J. C. Roder, A. A. Czitrom.
1990
. Class I (H-2Kb) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells.
Eur. J. Immunol.
20
:
841
15
McMillan, T. J., J. Rao, C. A. Everett, I. R. Hart.
1987
. Interferon-induced alterations in metastatic capacity, class I antigen expression and natural killer cell sensitivity of melanoma cells.
Int. J. Cancer
40
:
659
16
Storkus, W. J., R. D. Salter, P. Cresswell, J. R. Dawson.
1992
. Peptide-induced modulation of target cell sensitivity to natural killing.
J. Immunol.
149
:
1185
17
Brennan, J., D. Mager, W. Jefferies, F. Takei.
1994
. Expression of different members of the Ly-49 gene family defines distinct natural killer cell subsets and cell adhesion properties.
J. Exp. Med.
180
:
2287
18
Ortaldo, J. R., R. Winkler-Pickett, A. T. Mason, L. H. Mason.
1998
. The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells.
J. Immunol.
160
:
1158
19
Wang, L. L., I. K. Mehta, P. A. LeBlanc, W. M. Yokoyama.
1997
. Mouse natural killer cells express gp49B1, a structural homologue of human killer inhibitory receptors.
J. Immunol.
158
:
13
20
Y. Y., Yu., T. George, J. R. Dorfman, J. Roland, V. Kumar, M. Bennett.
1996
. The role of Ly-49A and 5E6 (Ly-49C) molecules in hybrid resistance mediated by murine natural killer cells against normal T cell blasts.
Immunity
4
:
67
21
Yokoyama, W. M., W. E. Seaman.
1993
. The Ly-49 and NKR-P1 gene families encoding lectin like receptors on natural killer cells: the NK gene complex.
Annu. Rev. Immunol.
11
:
613
22
Kane, K. P..
1994
. Ly-49 mediates EL4 lymphoma adhesion to isolated class I major histocompatibility complex molecules.
J. Exp. Med.
179
:
1011
23
Correa, I., D. H. Raulet.
1995
. Binding of diverse peptides to MHC class I molecules inhibits target cell lysis by activated natural killer cells.
Immunity
2
:
61
24
Itoh, T., W. J. Storkus, E. Gorelik, M. T. Lotze.
1994
. Partial purification of murine tumor-associated peptide epitopes common to histologically distinct tumors, melanoma and sarcoma, that are presented by H-2Kb molecules and recognized by CD8+ tumor-infiltrating lymphocytes.
J. Immunol.
153
:
1202
25
Holscher, M., A. L. Givan, C. G. Brooks.
1991
. The effect of transfected MHC class I genes on sensitivity to natural killer cells.
Immunology
73
:
44
26
Seaman, W. E., M. Sleisenger, E. Eriksson, G. C. Koo.
1987
. Depletion of natural killer cells in mice by monoclonal antibody to NK1. 1. Reduction in host defense against malignancy without loss of cellular or humoral immunity.
J. Immunol.
138
:
4539
27
Emoto, M., Y. Emoto, S. H. E. Kaufmann.
1995
. IL-4 producing CD4+ TCRαβint liver lymphocytes: influence of thymus, β2-microglobulin and NK1.1 expression.
Int. Immunol.
7
:
1729
28
Seo, N., K. Egawa.
1995
. Suppression of cytotoxic T lymphocyte activity by γ/δ T cells in tumor-bearing mice.
Cancer Immunol. Immunother.
40
:
358
29
Salvucci, O., F. Mami-Chouaib, J. L. Moreau, J. Theze, J. Chehimi, S. Chouaib.
1996
. Differential regulation of interleukin-12- and interleukin-15-induced natural killer cell activation by interleukin-4.
Eur. J. Immunol.
26
:
2736
30
Malnati, M. S., M. Peruzzi, K. C. Parker, W. E. Biddison, E. Ciccone, A. Moretta, E. O. Long.
1995
. Peptide specificity in the recognition of MHC class I by natural killer cell clones.
Science
267
:
1016
31
Peruzzi, M., N. Wagtmann, E. O. Long.
1996
. A p70 killer cell inhibitory receptor specific for several HLA-B allotypes discriminates among peptides bound to HLA-B*2705.
J. Exp. Med.
184
:
1585
32
Rajagopalan, S., E. O. Long.
1997
. The direct binding of a p58 killer cell inhibitory receptor to human histocompatibility leukocyte antigen (HLA)-Cw4 exhibits peptide selectivity.
J. Exp. Med.
185
:
1523
33
Dustin, M. L., T. A. Springer.
1989
. T cell receptor cross-linking transiently stimulates adhesiveness through LFA-1.
Nature
341
:
619
34
Matis, L. A., R. Cron, J. A. Bluestone.
1987
. Major histocompatibility complex-linked specificity of γδ receptor-bearing T lymphocytes.
Nature
330
:
262
35
Haregowoin, A., G. Soman, R. C. Hom, R. W. Finberg.
1989
. Human γδ T cells respond to mycobacterial heat-shock protein.
Nature
340
:
309
36
Porcelli, S., M. B. Brenner, J. L. Greenstein, S. P. Balk, C. Terhorst, P. A. Bleicher.
1989
. Recognition of cluster of differentiation 1 antigens by human CD4 CD8 cytolytic T lymphocytes.
Nature
341
:
447
37
Vidovic, D., M. Roglic, K. McKune, S. Guerder, C. Mckay, Z. Dembic.
1989
. Qa-1 restricted recognition of foreign antigen by γ/δ T cell hybridoma.
Nature
340
:
646
38
Ito, K., K. L. Van, M. Bonneville, S. Hsu, D. B. Murphy, S. Tonegawa.
1990
. Recognition of the product of a novel MHC TL region gene (27b) by a mouse γδ T cell receptor.
Cell
62
:
549
39
Hightower, L. E..
1991
. Heat shock, stress proteins, chaperones, and proteotoxicity.
Cell
66
:
191
40
Karlhofer, F. M., W. M. Yokoyama.
1991
. Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen: IL-2 activated NK cells possess additional specific stimulation pathways.
J. Immunol.
146
:
3662
41
Bezouska, K., C.-T. Yuen, J. O’Brien, R. A. Childs, W. Chai, A. M. Lawson, K. Drbal, A. Fiserova, M. Pospisil, T. Feizi.
1994
. Oligosaccharide ligands for NKR-P1 protein activate NK cells and cytotoxicity.
Nature
372
:
150
42
Ryan, J. C., E. C. Niemi, M. C. Nakamura, W. E. Seaman.
1995
. NKR-P1A is a target-specific receptor that activates natural killer cell cytotoxicity.
J. Exp. Med.
181
:
1911
43
Berke, G..
1995
. Unlocking the secrets of CTL and NK cells.
Immunol. Today
16
:
343
44
Arase, H., N. Arase, Y. Kobayashi, Y. Nishimura, S. Yonehara, K. Onoe.
1994
. Cytotoxicity of fresh NK1.1+ T cell receptor α/β+ thymocytes against a CD4+ 8+ thymocyte population associated with intact Fas antigen expression on the target.
J. Exp. Med.
180
:
423
45
Martiniello, R., R. C. Burton, Y. C. Smart.
1997
. Natural cell-mediated cytotoxicity (NCMC) against NK-sensitive tumors in vitro by murine spleen Ly-6C+ natural T cells.
Int. J. Cancer
70
:
450
46
Blumberg, R. S., C. Terhorst, P. Bleicher, F. V. McDermott, C. H. Allan, S. B. Landau, J. S. Trier, S. P. Balk.
1991
. Expression of a nonpolymorphic MHC class I-like molecule, CD1D, by human intestinal epithelial cells.
J. Immunol.
147
:
2518
47
Bendelac, A., O. Lantz, M. E. Quimby, J. W. Yewdell, J. R. Bennink, R. R. Brutkiewicz.
1995
. CD1 recognition by mouse NK1+ T lymphocytes.
Science
268
:
863
48
Kikly, K., G. Dennert.
1992
. Evidence for a role for T cell receptors (TCR) in the effector phase of acute bone marrow graft rejection.
J. Immunol.
149
:
3489
49
McMenamin, C., C. Pimm, M. McKersey, P. G. Holt.
1994
. Regulation of IgE responses to inhaled antigen in mice by antigen-specific γδ T cells.
Science
265
:
1869
50
Gorczynski, R. M..
1994
. Adoptive transfer of unresponsiveness to allogeneic skin grafts with hepatic γδ+ T cells.
Immunology
81
:
27
51
Szczepanik, M., L. R. Anderson, H. Ushio, W. Ptak, M. J. Owen, A. C. Hayday, W. Askenase.
1996
. γδ T cells from tolerized αβ T cell receptor (TCR)-deficient mice inhibit contact sensitivity-effector T cells in vivo, and their interferon-γ production in vitro.
J. Exp. Med.
184
:
2129
52
Kaminski, M. J., P. D. Cruz, P. R. Bergstresser, Jr, A. Takashima.
1993
. Killing of skin-derived tumor cells by mouse dendritic epidermal T-cells.
Cancer Res.
53
:
4014
53
Borm, W., L. Hall, A. Dallas, J. Boymel, T. Shinnick, D. Young, P. Brennan, R. O’Brien.
1990
. Recognition of peptide antigen by heat-shock reactive γδ T lymphocytes.
Science
249
:
67
54
Weintraub, B. C., M. R. Jackson, S. M. Hedric.
1994
. γδ T cells can recognize nonclassical MHC in the absence of conventional antigenic peptides.
J. Immunol.
153
:
3051
55
Kim, H. T., E. L. Nelson, C. Clayberger, M. Sanjanwala, J. Sklar, A. M. Krensky.
1995
. γδ T cell recognition of tumor Ig peptide.
J. Immunol.
154
:
1614