HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seo, N. Articles by Takigawa, M. Search for Related Content PubMed PubMed Citation Articles by Seo, N. Articles by Takigawa, M.

Down-Regulation of Tumoricidal NK and NK T Cell Activities by MHC K^b Molecules Expressed on Th2-Type T and ß T Cells Coinfiltrating in Early B16 Melanoma Lesions

Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined whether T and ß 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-, whereas T and a large part of ß 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. T and ß T cell populations dramatically inhibited the cytotoxicity of NK and NK T cells in an MHC K^b-dependent manner. Culture supernatant from T and ß 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 T cell-depleted mice significantly lysed B16 cells compared with those from control mice. Finally, anti-K^b 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 T and ß 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has been well known that several kinds of lymphocytes, e.g., NK, NK1.1⁺ ßTCR⁺ T (NK T), T, and conventional ß 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- 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- (10). T cells can be classified into Th1 and Th2 types as well as ß 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- 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-2K^b molecules derived from the H-2^b allele (20). However, it remains unknown whether Ly49C can interact with the D^b molecule. Ly49A is expressed on certain subsets of B6 mouse (H-2^b) NK cells (21), but not on NK cells derived from H-2^d or H-2^k mice. Affinity-purified K^b and D^b molecules, however, bind to Ly49A with low affinity (22), suggesting that relevant peptide-K^b or -D^b 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 K^b expression is undetectable on the surface of B16 cells. Our results show that T and ß T cells that predominantly coinfiltrate in the tumor lesions inhibit the tumoricidal activities of NK and Th1-type NK T cells in a K^b-dependent manner and suppress the proliferation of these NK-lineage cells by releasing soluble factors, including Th2 cytokines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and tumor cells

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).

Monoclonal Abs

The following mAbs were purchased from PharMingen (San Diego, CA): FITC-conjugated anti-NK1.1 (PK136), anti-H-2D^b (KH95), and anti-H-2K^b (AF6-88.5); phycoerythrin (PE)-conjugated anti-ßTCR (H57-597) and anti-TCR (GL3); purified form of anti-ßTCR (H57-597); anti-TCR (GL3); anti-H-2K^b (AF6-88.5); anti-H-2D^b (KH95); anti-CD3 (145-2C11); anti-CD16/CD32 (2.4G2); rat IgG2b mAb (R35-38); anti-IFN- (R4-6A2); anti-IL-4 (BVD4-1D11) and anti-IL-10 (JES5-2A5) mAbs; and biotin-conjugated anti-IFN- (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-2K^b and anti-H-2D^b mAbs were prepared using the ImmunoPure Fab preparation kit (Pierce, Rockford, IL) on the basis of papain cleavage of Igs. Hybridoma producing anti-TCR 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.

Preparation of subpopulations of TILs and normal splenic NK1.1⁺ cells

RPMI 1640 (Nissui, Tokyo, Japan) medium supplemented with 25 mM HEPES, 2 mM L-glutamine, 1 mM nonessential amino acid, 5 x 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 x 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 x 10⁵ cells/ml in PBS containing 10% FCS) applied on 5 ml of Histopaque 1083 were subjected to centrifugation at 1000 x 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-ßTCR (H57–597), anti-TCR (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 x 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 x 10⁵, 62 and 36% populations of the cells were NK and NK T cells, respectively, as assessed by flow cytometry). ß T and T cell populations were further separated from the NK1.1⁺ cell-depleted 5-day TILs. The cells (4 x 10⁵) were incubated with anti-ßTCR 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 x 10⁵) were incubated with anti-TCR 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 (ß T cells, 1 x 10⁵; T cells, 1 x 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, 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-ßTCR mAb-conjugated beads and used as cultured NK T cells (NK1.1⁺ ßTCR⁺, 98% purity). The remaining cells were used as cultured NK cells (NK1.1⁺ ßTCR^- TCR^-, 84% purity). Cultured ß T and T cells were prepared from 5-day B16 TILs as follows. Anti-ßTCR mAb (H57-597)- or anti-TCR 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-ßTCR mAb- or anti-TCR mAb-coated plates (1 x 10⁵ cells/well) for 5 days. The expanded cells were harvested and used as ß T (NK1.1^-, ßTCR⁺ TCR^-; 99% purity) and T (NK1.1^-, ßTCR^- TCR⁺; 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 x 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⁺ ßTCR^-, 83%; NK1.1⁺ ßTCR⁺, 10%).

Flow cytometric analysis

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 x g, 30 min), and pelleted cells were collected and stained with FITC-conjugated anti-K^b, anti-D^b, 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.

ELISPOT assay

The cytokine profiles of NK, NK T, ß T, and 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- (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 x 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- (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.

In vivo Ab treatments

To test class I-dependent inhibition of NK cells at tumor sites, 100 µg (in 50 µl of PBS)/mouse of anti-H-2K^b Fab mAb, anti-H-2D^b 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 T cell-depleted B6 mice, 500 µg/mouse of anti-TCR (UC7-13D5) or hamster IgG Ab as a control was administered i.v. to B6 mice. The disappearance of TCR⁺ 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 x 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.

Cytotoxicity assay

Varying numbers of separated TIL populations, NK, NK T, ß T, or T cells, were assayed by incubation with 1 x 10^{4 51}Cr-labeled freshly isolated or cultured B16 or YAC-1 for 5 h at 37°C. Otherwise, the cytotoxicities of separated ß T and T cells were examined by incubation with 1 x 10^{4 51}Cr-labeled cultured NK or NK T cells for 5 h at 37°C. Target cells were radiolabeled by suspension at a concentration of 1 x 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 T or ß T cells on NK and NK T cell cytotoxicities against B16 and YAC-1 were tested by the addition of freshly isolated or cultured T or ß T cells to the cytotoxicity assay in varying numbers.

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

Proliferation assay

The cultured NK and NK T cells (2 x 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 T or ß T cells prepared as described above were added to the NK or NK T cell proliferation assay in varying volumes.

Treatment of cells with anti-class I mAb

To determine class I-dependent ß T or T cell inhibition of NK or NK T cell cytotoxicity, ß T or T cells were preincubated with K^b- and D^b-specific Fab mAb or control mouse IgG Fab Ab (10 µg/ml). After washing three times with DMEM, the Ab-treated ß T or 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 K^b- and D^b-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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
NK, NK T, T, and ß T cells predominantly infiltrate in 5-day B16 tumors

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 1A, NK (NK1.1⁺, ßTCR^-), NK T (NK1.1⁺, ßTCR⁺), ß T (NK1.1^-, ßTCR⁺), and T cells (NK1.1^-, TCR⁺) were present at 32, 21, 11, and 17%, respectively, of the total 5-day TILs. A small number of NK1.1⁺ TCR⁺ 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. 1B). Therefore, NK, NK T, ß T, and T cells transiently accumulated soon after tumor cell implantation.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Accumulation of NK, NK T, ß T, and T cells at the early stage of B16 development. A, Freshly isolated 5-day B16 TILs were double stained with PE-conjugated anti-ßTCR or anti-TCR 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-ßTCR or anti-TCR 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+, ßTCR+, and TCR+ cells in the B16 tumor suspension. PE-conjugated mouse IgG () and hamster IgG () were used as controls.

We examined whether each of four 5-day TIL populations has the ability to lyse B16 cells. Short term cultured (C-) NK, NK T, ß T, and T populations or freshly isolated (F-) NK1.1⁺, ß T, and 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. 2A). 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-ß T and C- and F- T cells had no cytotoxicity against any tumor target.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Cytotoxicity of TILs and NK, NK T, ß T, and T cell populations. A, Short term 5-day C-B16 tumor-infiltrating NK, NK T, ß T, and 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+, ß T, and 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.

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.

Inhibition of the cytotoxicity and proliferation of NK and NK T cells by T and ß T 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 ß T or T cells. NK and NK T cell cytotoxicities against C-B16 and YAC-1, respectively, were dose dependently decreased by the ß T cell or T cell population, although the inhibitory effect of T cells was stronger than that of ß T cells (Fig. 3A). The NK cell cytotoxicity was absolutely abrogated at a T cell/NK cell ratio of 1 (5 x 10⁴/well). This inhibition was not due to direct killing because neither T nor ß T cell populations lysed NK or NK T cells as assessed by cytotoxic assay against ⁵¹Cr-labeled NK or NK T cells (Fig. 3B). The proliferation of NK and NK T cell populations was quickly decreased after coculture with the ß T or T cell population (Fig. 3C). Again, the suppressive ability of T cells was higher than that of ß T cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Inhibitory effects of 5-day B16 tumor-infiltrating ß T and T cells on the cytotoxicity and proliferation of NK and NK T cells. A, The short term cultured ß T or 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 ß T and 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 x 105 cells/well) were cocultured with the indicated numbers of mitomycin C-treated ß T or 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.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Enhanced anti-B16 cytotoxicity of 5-day B16 TILs freshly isolated from T cell-depleted mice. Fresh 5-day B16 TILs were separated from T-depleted or control hamster IgG-treated mice. NK1.1+ cell-removed fractions of TILs from 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.

Culture supernatants from Th2-type T and ß T cells suppress the proliferation of NK and NK T cells, but not their cytotoxicities

Fresh NK1.1⁺, ß T, and 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-, and only a few of them (3.7% of the total number) had the ability to produce IL-4 and IL-10 (Fig. 5A). On the contrary, ß T and T cell populations secreted IL-4 and IL-10 at high levels, with 8.9% of the total ß T cell number weakly expressing IFN-. Furthermore, short term cultivation of each population did not influence on its cytokine profile (NK: IL-4, 0%; IFN-, 88%; NK T: IL-4, 2.8%; IFN-, 72%; ß T: IL-4, 65%; IFN-, 14%; T: IL-4, 83%; IFN-, 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--producing cells was small (3.2%; Fig. 5B). This raised the possibility that Th2 cytokine-producing ß T and T cells inhibit Th1-type NK and NK T cell activities by releasing cytokines. Therefore, the modulatory effects of T and ß 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 T or ß 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 T and ß T cells are only partly involved in the mechanisms by which they inhibit NK and NK T cell cytotoxicity.

View larger version (18K): [in this window] [in a new window]   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+, ß T or 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--producing cells (5 x 103 in duplicate) were enumerated by ELISPOT assay. B, Five-day F-B16 TILs (5 x 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.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Inhibition of the proliferation, but not the cytotoxicity, of NK and NK T cell populations by culture supernatants from T and ß T cell populations. Culture supernatants of short term cultured T or ß 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.

Inhibition of NK and NK T cell cytotoxicities by T and ß T cells is dependent on class I molecules

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

View larger version (43K): [in this window] [in a new window]   FIGURE 7. Inhibition of NK and NK T cell cytotoxicity by ß T and T cells in a Kb-dependent fashion. A, An anti-Kb- or anti-Db-treated or control mouse IgG-treated, cultured T or ß 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 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.

Tumor growth inhibition by administration of K^b-specific Fab mAb in B16 melanoma lesions

On the basis of the concept that T and ß T cells exert an anti-NK and NK T cell action with their K^b molecules, we had anticipated that administration of K^b-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). K^b- or D^b-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 K^b-specific mAb that was injected consecutively on days 4 to 6 after tumor inoculation, whereas the growth was not affected by D^b-specific or control mAb (Fig. 8). However, administration of anti-K^b mAb on days 12 to 14 failed to inhibit B16 growth (data not shown). This finding is in accordance with the observation that T and ß T cells accumulated in the B16 lesion markedly 5 to 7 days after tumor inoculation (see Fig. 1B). In contrast, K^b-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 K^b molecules on T and ß T cells in the inhibition of NK and NK T cells.

View larger version (24K): [in this window] [in a new window]   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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has been shown that NK cells is a major IFN- 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- 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 T and ß 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 K^b expression. Instead, T and ß T cells that accumulated in B16 tumor lesions down-modulated the cytotoxicity of NK cells. This down-regulation was restricted by K^b molecules expressed on these coinfiltrating T cells. In addition, our study demonstrated that activated, but not resting, splenic T cells from naive mice inhibited NK cell activity in a K^b-dependent manner. One possible explanation for these findings is that some endogenous peptides synthesized in activated T and ß T cells can inhibit NK and NK T cell activities when presented in the context of the K^b 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 K^b gene-transfected NK-sensitive tumor cells that conjunctively express peptides acquire resistance against NK cell attack (14, 15). Although, the binding affinity of purified K^b molecule to Ly49 is too low to negatively regulate Ly49⁺ NK cells (22), K^b-restricted peptides induced in activated T and ß T cells are possibly able to alter the K^b 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 K^b molecules on T and ß 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 K^b molecules without endogenous peptides.

It remains to be elucidated whether the T and ß T cells accumulating in early B16 tumor lesions recognize B16 cells. TCRs can recognize not only classical MHC class I complexes (34) in the same way as target recognition by ßTCR 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 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 ßTCR 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 ßTCR, in tumor lesions, because, as indicated in this study, the NK T cell population isolated from B16 TILs secreted IFN-, but not IL-4 or IL-10, as shown in a cytokine production pattern induced by NK1.1 ligation (10).

Immunoregulatory roles for T cells have been well studied in various systems (28, 49, 50, 51). T cells seem to act as suppressors of cytotoxic cells in tumor environments. T cells purified from tumor-bearing mice inhibit CTL activities through an unknown factor(s) (28). In addition, our study provides evidence that T cells, via their K^b molecules, block the cytotoxicity of NK and NK T cells in the B16-bearing animal model. Of interest is the observation that these 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, 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 T cells may be classified into the cytotoxic effector and the immunoregulator, respectively.

	Acknowledgments

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

	Footnotes

 
¹ This work was supported in part by a grant from the Ministry of Health of Japan and by the Shiseido fund.

² Address correspondence and reprint requests to Dr. Naohiro Seo, Department of Dermatology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan. E-mail address:

³ Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; PE, phycoerythrin; ELISPOT, enzyme-linked immunospot; C-, cultured; F-, freshly isolated.

Received for publication February 2, 1998. Accepted for publication June 9, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kowalczyk, D., W. Skorupski, Z. Kwias, J. Nowak. 1996. Activated / T lymphocytes infiltrating renal cell carcinoma. Immunol. Lett. 53:15.[Medline]
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.[Abstract]
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.[Medline]
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.[Abstract]
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.[Abstract]
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.[Medline]
7. Scharton, T. M., P. Scott. 1993. Natural killer cells are a source of interferon- that derives differentiation of CD4⁺ T cell subsets and induces early resistance to Leishmania major in mice. J. Exp. Med. 178:567.[Abstract/Free Full Text]
8. Trinchieri, G.. 1994. Recognition of major histocompatibility complex class I antigens by natural killer cells. J. Exp. Med. 180:417.[Free Full Text]
9. Yoshimoto, T., W. E. Paul. 1994. CD4^pos, NK1.1^pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J. Exp. Med. 179:1285.[Abstract/Free Full Text]
10. Arase, H., N. Arase, T. Saito. 1996. Interferon- production by natural killer cells and NK1.1⁺ T cells upon NKR-P1 cross-linking. J. Exp. Med. 183:2391.[Abstract/Free Full Text]
11. Wen, L., D. F. Barber, W. Pao, F. S. Wong, M. J. Owen, A. Hayday. 1998. Primary 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.[Abstract/Free Full Text]
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.[Medline]
13. Kesari, V. K., J. Geliebter. 1993. Rejuvenated expression of H-2K^b in RMA-S cells does not affect alloreactive T cell- and natural killer cell-mediated lysis. Immunol. Lett. 38:77.[Medline]
14. Carlow, D. A., U. Payne, N. Hozumi, J. C. Roder, A. A. Czitrom. 1990. Class I (H-2K^b) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells. Eur. J. Immunol. 20:841.[Medline]
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.[Medline]
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.[Abstract]
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.[Abstract/Free Full Text]
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.[Abstract/Free Full Text]
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.[Abstract]
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.[Medline]
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.[Medline]
22. Kane, K. P.. 1994. Ly-49 mediates EL4 lymphoma adhesion to isolated class I major histocompatibility complex molecules. J. Exp. Med. 179:1011.[Abstract/Free Full Text]
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.[Medline]
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-2K^b molecules and recognized by CD8⁺ tumor-infiltrating lymphocytes. J. Immunol. 153:1202.[Abstract]
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.[Medline]
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.[Abstract]
27. Emoto, M., Y. Emoto, S. H. E. Kaufmann. 1995. IL-4 producing CD4⁺ TCRß^int liver lymphocytes: influence of thymus, ß₂-microglobulin and NK1.1 expression. Int. Immunol. 7:1729.[Abstract/Free Full Text]
28. Seo, N., K. Egawa. 1995. Suppression of cytotoxic T lymphocyte activity by / T cells in tumor-bearing mice. Cancer Immunol. Immunother. 40:358.[Medline]
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.[Medline]
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.[Abstract/Free Full Text]
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.[Abstract/Free Full Text]
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.[Abstract/Free Full Text]
33. Dustin, M. L., T. A. Springer. 1989. T cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341:619.[Medline]
34. Matis, L. A., R. Cron, J. A. Bluestone. 1987. Major histocompatibility complex-linked specificity of receptor-bearing T lymphocytes. Nature 330:262.[Medline]
35. Haregowoin, A., G. Soman, R. C. Hom, R. W. Finberg. 1989. Human T cells respond to mycobacterial heat-shock protein. Nature 340:309.[Medline]
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.[Medline]
37. Vidovic, D., M. Roglic, K. McKune, S. Guerder, C. Mckay, Z. Dembic. 1989. Qa-1 restricted recognition of foreign antigen by / T cell hybridoma. Nature 340:646.[Medline]
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 T cell receptor. Cell 62:549.[Medline]
39. Hightower, L. E.. 1991. Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66:191.[Medline]
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.[Abstract]
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.[Medline]
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.[Abstract/Free Full Text]
43. Berke, G.. 1995. Unlocking the secrets of CTL and NK cells. Immunol. Today 16:343.[Medline]
44. Arase, H., N. Arase, Y. Kobayashi, Y. Nishimura, S. Yonehara, K. Onoe. 1994. Cytotoxicity of fresh NK1.1⁺ T cell receptor /ß⁺ thymocytes against a CD4⁺ 8⁺ thymocyte population associated with intact Fas antigen expression on the target. J. Exp. Med. 180:423.[Abstract/Free Full Text]
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.[Medline]
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.[Abstract/Free Full Text]
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.[Abstract/Free Full Text]
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.[Abstract]
49. McMenamin, C., C. Pimm, M. McKersey, P. G. Holt. 1994. Regulation of IgE responses to inhaled antigen in mice by antigen-specific T cells. Science 265:1869.[Abstract/Free Full Text]
50. Gorczynski, R. M.. 1994. Adoptive transfer of unresponsiveness to allogeneic skin grafts with hepatic ⁺ T cells. Immunology 81:27.[Medline]
51. Szczepanik, M., L. R. Anderson, H. Ushio, W. Ptak, M. J. Owen, A. C. Hayday, W. Askenase. 1996. T cells from tolerized ß T cell receptor (TCR)-deficient mice inhibit contact sensitivity-effector T cells in vivo, and their interferon- production in vitro. J. Exp. Med. 184:2129.[Abstract/Free Full Text]
52. Kaminski, M. J., P. D. Cruz, Jr P. R. Bergstresser, A. Takashima. 1993. Killing of skin-derived tumor cells by mouse dendritic epidermal T-cells. Cancer Res. 53:4014.[Abstract/Free Full Text]
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 T lymphocytes. Science 249:67.[Abstract/Free Full Text]
54. Weintraub, B. C., M. R. Jackson, S. M. Hedric. 1994. T cells can recognize nonclassical MHC in the absence of conventional antigenic peptides. J. Immunol. 153:3051.[Abstract]
55. Kim, H. T., E. L. Nelson, C. Clayberger, M. Sanjanwala, J. Sklar, A. M. Krensky. 1995. T cell recognition of tumor Ig peptide. J. Immunol. 154:1614.[Abstract]

This article has been cited by other articles:

				H. M. Ashour and J. Y. Niederkorn {gamma}{delta} T Cells Promote Anterior Chamber-Associated Immune Deviation and Immune Privilege through Their Production of IL-10 J. Immunol., December 15, 2006; 177(12): 8331 - 8337. [Abstract] [Full Text] [PDF]

				B. Wu, A. Ootani, R. Iwakiri, Y. Sakata, T. Fujise, S. Amemori, F. Yokoyama, S. Tsunada, S. Toda, and K. Fujimoto T Cell Deficiency Leads to Liver Carcinogenesis in Azoxymethane-Treated Rats Experimental Biology and Medicine, January 1, 2006; 231(1): 91 - 98. [Abstract] [Full Text] [PDF]

				C. Dosiou and L. C. Giudice Natural Killer Cells in Pregnancy and Recurrent Pregnancy Loss: Endocrine and Immunologic Perspectives Endocr. Rev., February 1, 2005; 26(1): 44 - 62. [Abstract] [Full Text] [PDF]

				E. Y. Chiang, M. Henson, and I. Stroynowski Correction of Defects Responsible for Impaired Qa-2 Class Ib MHC Expression on Melanoma Cells Protects Mice from Tumor Growth J. Immunol., May 1, 2003; 170(9): 4515 - 4523. [Abstract] [Full Text] [PDF]

				H. Ota, H. Rong, S. Igarashi, and T. Tanaka Suppression of natural killer cell activity by splenocyte transplantation in a rat model of endometriosis Hum. Reprod., June 1, 2002; 17(6): 1453 - 1458. [Abstract] [Full Text] [PDF]

				M. E. Skelsey, J. Mellon, and J. Y. Niederkorn {{gamma}}{{delta}}T Cells Are Needed for Ocular Immune Privilege and Corneal Graft Survival J. Immunol., April 1, 2001; 166(7): 4327 - 4333. [Abstract] [Full Text] [PDF]

				J. Pinilla-Ibarz, K. Cathcart, T. Korontsvit, S. Soignet, M. Bocchia, J. Caggiano, L. Lai, J. Jimenez, J. Kolitz, and D. A. Scheinberg Vaccination of patients with chronic myelogenous leukemia with bcr-abl oncogene breakpoint fusion peptides generates specific immune responses Blood, March 1, 2000; 95(5): 1781 - 1787. [Abstract] [Full Text] [PDF]

				N. Seo, Y. Tokura, M. Takigawa, and K. Egawa Depletion of IL-10- and TGF-{beta}-Producing Regulatory {gamma}{delta} T Cells by Administering a Daunomycin-Conjugated Specific Monoclonal Antibody in Early Tumor Lesions Augments the Activity of CTLs and NK Cells J. Immunol., July 1, 1999; 163(1): 242 - 249. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seo, N. Articles by Takigawa, M. Search for Related Content PubMed PubMed Citation Articles by Seo, N. Articles by Takigawa, M.