NKG2D engagement of colorectal cancer-specific T cells strengthens TCR-mediated antigen stimulation and elicits TCR independent anti-tumor activity

Authors


Abstract

The NKG2D receptor is expressed by human NK, γ δ T and α/β T lymphocytes and its engagement results in the stimulation of effector cells. We evaluated the role of NKG2D receptor in anti-colorectal cancer (CRC) immune response. The cell surface expression of stress-inducible NKG2D ligands MICA/B (MHC class I-related chain molecules A/B) and ULBP (UL16 binding protein) by apanel of CRC lines was evaluated by flow cytometry. MICA and ULBP2/3 were widely expressed by the analyzed lines, with a minority of them being also ULBP-1+, whereas MICB was undetectable.CD8+ and CD4+ HLA-restricted anti-tumor T cell clones of a CRC patient were used to evaluate whether NKG2D engagement could mediate tumor recognition. Three out of four CD8+ T cell clones recognized the autologous tumor with a marginal NKG2D engagement, a finding that was correlated with the weak expression of NKG2D ligands by the autologous tumor. On the contrary, NKG2D triggering of these CD8+ T cell clones induced recognition of allogeneic CRC lines showing high expression of MICA and ULBP. A costimulatory role of NKG2D was observed with one CD4+/NKG2D+ T cell clone when stimulated by tumors sharing the HLA class II alleles and expressing NKG2D ligands. Taken together these data indicate that the engagement of NKG2D, depending on the expression of its ligands by target cells, can influence the pattern of anti-tumor reactivity by T lymphocytes.

Abbreviations:
CRC:

Colorectal cancer

MIC:

MHC class I-related chain molecules

MFI:

Mean fluorescence intensity

TAA:

Tumor-associated antigen

ULBP:

UL16 binding protein

1 Introduction

T lymphocytes recognize tumor-associated antigens (TAA) as peptides in association with HLA molecules on the cell surface of tumor cells. A growing number of TAA have been characterized by theisolation of anti-tumor T cells 1, although the complexity of T cell-mediated anti-tumor response has not been completely dissected. Stress-inducible MHC class I-related molecules, MICA, MICB and UL16 binding protein (ULBP), have been shown to be constitutively expressed by the gastrointestinal epithelium and to be induced upon stress signals in fibroblasts, endothelial anddendritic cells 2, 3. Furthermore, these molecules are expressed by epithelial tumors 47 and, as recently shown, by melanomacells 810. MICA/B and ULBP are ligands of the NKG2D receptor, which is expressed by NK, α/β+T and γ/δ+ T lymphocytes 3, 11. Through the engagement of NKG2D, human NK and γ/δ+ T lymphocytes lyse tumor cells expressing MICA; the lysis is inhibited following incubation of the target cells with specific mAb directed to MICA or NKG2D 4, 5, 12. Moreover, NKG2D-mediated recognition of non-epithelial human tumors by NK 8, 10 and its role as costimulatory signal, following the triggering of TCR, in α/β+ T cells 13 has been demonstrated.Furthermore, for γ/δ T cells that recognize stress-treated epithelial tissue, NKG2D ligands deliver both TCR- and NKG2D-dependent costimulatory signals 14. The role of NKG2D in anti-tumor activity has been demonstrated by the evidence that the ectopic expression of the murine NKG2D ligands leads to the in vivo rejection of tumors by NK and/or CD8+ T cells 15. Although the regulation of stimulatory and costimulatory signals engaged in T cell-mediated anti-tumor response has not been conclusively assessed, the available data underline the need to further investigate the role of NKG2D in the immune response to tumors. This idea is also supported by the evidence that NKG2D+ cells, either CD3+ or CD56+, have been found in the tumor-infiltrating lymphocytes of human melanoma patients, although the anti-tumor effector function of these lymphocytes was not investigated 9.

The aim of the present study was to see whether the anti-tumor activity of T lymphocytes isolated from colorectal cancer (CRC) patients is dependent on the engagement of NKG2D receptors. To this purpose the expression of NKG2D ligands MICA, MICB and ULBP has been evaluated in a panel of human CRC lines, including some fresh in vitro established ones. Both CD8+ and CD4+ T lymphocyte clones from PBMC of a CRC patient showing a broad spectrum of anti-tumor reactivity have been isolated and their differentially regulated usage of TCR and NKG2D in anti-tumor reactivity was studied. Our data show that different T lymphocyte-mediated anti-tumor reactivity was achieved depending on the engagement of TCR and NKG2D, which in turns depends on the expression of its ligand by tumor cells. These findings provide the basis for further investigation of the role of the NKG2D-driven T cell-mediated tumor recognition and its significance in tumor immunosurveillance.

2 Results

2.1 Expression of NKG2D ligands by colorectal tumor lines

The surface expression of MICA, MICB, ULBP1, 2 and 3 have been evaluated by indirect immunofluorescence and FACScan analysis. As shown in Table 1, three out of seven CRC lines expressed high levels of MICA (124–280 mean fluorescence intensity, MFI); the 1847 col line showed an intermediate expression (93 MFI and 54% of positive cells), whereas 1869 col and 1870 col were defective for MICA along with the LoVo line that is known to lack β2-microglobulin and class I HLA 16. The analyzed lines failed to express cell surface MICB molecules, despite detection of the MICB mRNA by reverse transcription-PCR analysis (data not shown). Failure in the expression of ULBP1 was observed in five out of seven CRC lines, while 1870 col and SW480 were weakly positive for this molecule (Table 1). ULBP2 was expressed with high MFI by 23–31% of 1869 col, 1870 col and 1872 col cells, whereas ULBP3 was highly expressed only by 1847 col. As control, the melanoma line Me1681 showed high expression of MICA only. No significant expression of the NKG2D ligands was observed on the 1869 EBV-B.

These data indicate that a heterogeneous expression of NKG2D ligands occurs in tumor lines, and that ULBP molecules can be expressed, although not homogeneously, by CRC lines even in absence of MIC molecules.

Table 1. Expression of NKG2D ligands by tumor cell lines evaluated by immunofluorescence and cytofluorimetric analysisa)
  1. a) The expression of MICA, MICB, ULBP-1, ULBP-2 and ULBP-3 was evaluated with the following mAb, respectively: BAM 195, M362, M295, M310, and M551. Values represent MFI of the positive cells with background obtained from the negative control subtracted. Bold values represent significant MFI (>40). Number in parenthesis represents the percentage of positive cells. n.d.: not done.

  2. b) EBV-transformed B cells.

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2.2 Isolation of anti-tumor T cell clones from the 1869 PBMC

Tumor-specific T lymphocyte clones were generated by limiting dilution of PBMC isolated from the CRC patient 1869 and cultured in vitro with irradiated autologous tumor cells. The T cell clones M21, M23, M51 and M83 showed class I HLA-restricted recognition of the autologous 1869 col cells (Table 2). In contrast, clone M26 and the previously isolated CD4+ T cell clone C111 that recognizes a new CRC-associated antigen (COA-1) (Maccalli et al., manuscript in preparation), reacted against the autologous tumor in an MHC class II-restricted fashion, since inhibition of IFN-γ secretion occurred following treatment of the tumor target cells with the L243 but not with W6/32 mAb. The same effectors did not recognize the autologous EBV-lymphoblastoid B cells (except for a weak reactivity of the M26 and C111 clones), and CD40 ligand (CD40L)-cultured normal B cells (1872 and 1847 CD40L-B) (Table 2). The phenotype analysis of the T cell clones showed that M21, M23, M51 and M83 expressed homogeneously CD3, CD8, TCR α/β, NKG2D and CD56; C111 expressed CD3, CD4, TCR α/β, NKG2D and CD56; while M26 was CD3+, CD4+, TCR α/β+, NKG2D and CD56+ (data not shown).

Table 2. Anti-CRC lines reactivity by T cell clones isolated from PBMC of patient 1869a)
  1. a) T cells (1.5×104) were incubated with target cells (2×104) in 96-well plate in 250-μl final volume of AIMV plus 2% HS. Supernatants were collected after 18 h and IFN-γ content evaluated. The tumor cell lines were pre-incubated with 3,000 IU rhIFN-γ. Values represent pg/ml of IFN-γ. Significant cytokine release (taken as >100 pg/ml, underlined values) determined by statistical analysis of differences between the means of IFN-γ released by T cells alone vs. T cell incubated with target cells (p<0.01). Bold values represent significant (p<0.01) inhibition of cytokine release evaluated by statistical analysis of differences between the means of IFN-γ released by T cells incubated with tumor cell lines vs. T cell incubated with tumor cells pre-exposed to mAb directed to HLA class I or class II. SD for duplicate determination was <10% of the mean.

  2. b) Target cells pre-incubated with anti-HLA class I mAb W6/32.

  3. c) Target cells pre-incubated with anti-HLA-DR mAb L243.

  4. d) EBV-transformed B cells.

  5. e) B cells cultured with 100 ng/ml CD40L and 100 IU/ml IL-4.

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2.3 The engagement of both the TCR and NKG2D occurs in CD8+ T cell clones exhibiting anti-tumor reactivity

2.3.1 Involvement of NKG2D in CD8+ T cell clone-mediated tumor recognition

The anti-tumor activity of the CD8+ T lymphocyte clones was further characterized on autologous and allogeneic CRC target lines. Furthermore, we investigated whether the NKG2D receptor, expressed by these effectors, could mediate the anti-tumor recognition.

Fig. 1 shows that, after stimulation with the autologous 1869-B7–1 col line, M51 T cells released IFN-γ, a reaction that was inhibited (70%) by the anti-HLA class I mAb (W6/32) and by pre-incubating T lymphocytes with the anti-TCR mAb (75%), indicating that these lymphocytes recognized a defined antigen presented in association with class I HLA on the surface of autologous tumor cells. Furthermore, a weak but statistically significant inhibition (33%, p≤0.01) of IFN-γ release was observed when M51 cells were pre-incubated with the anti-NKG2D mAb. These data indicate that the specific reactivity of M51 T cells was mainly exerted by the triggering of the TCR, although a minor role could have been played by NKG2D stimulation. These results have been confirmed by the weak synergic blocking of TCR and NKG2D after pre-incubation of T cells with both specific mAb (Fig. 1) that correlates with the weak expression of NKG2D ligands by the 1869 col line (see Table 1). Thus, limited availability of these molecules on tumor cells might be responsible of the weak effect of engagement of NKG2D.

Significant (p≤0.01) IFN-γ secretion by M51 lymphocytes was observed even in the presence of the allogeneic CRC lines 1872 col and 1847 col though incubation with anti-HLA class I (W6/32) or anti-TCR mAb did not affect cytokine release (Fig. 1, gray and black bars). T cell stimulation by these tumor lines was inhibited after incubation of the effectors with the anti-NKG2D mAb (60% and 48% inhibition with 1872 col and 1847 col, respectively). No additional inhibition of cytokine release was observed after a simultaneous blocking of TCR and NKG2D, indicating that the allogeneic tumor recognition was only mediated by the engagement of NKG2D. Lack of reactivity on the class I HLA and NKG2D ligand negative LoVo line 16 (see also Table 1), supports the conclusion that clone M51 exerted TCR- and NKG2D-mediated anti-tumor activity.

NKG2D-mediated tumor recognition was observed also for M21 and M23 clones (Fig. 2A and B, respectively). IFN-γ was released after stimulation of these CD8+/ NKG2D+ T lymphocytes by the autologous tumor, although a weak inhibition (25% and 31% for M21 and M23, respectively) followed the incubation of the targets with the anti-HLA class I mAb. A stronger inhibition was achieved following incubation of these effectors with anti-NKG2D mAb (44% and 45% for M21 and M23, respectively) (Fig. 2A, B white columns). These T cell clones, however, showed an HLA-unrestricted recognition of the allogeneic CRC line 1847 col, whereas pre-incubation of these lymphocytes with the anti-NKG2D mAb inhibited the reaction (50% and 55% for clone M21 and M23, respectively) (Fig. 2A, B black columns). Reactivity by these clones against the allogeneic tumor line 1872 col was also observed and this tumor recognition was weakly but significantly inhibited by the anti-HLA class I mAb for M21 (33%) but not for M23. This finding may be explained by the recognition of an HLA-A3-restricted TAA by M21 T cells since this allele was shared between the 1872 col and the autologous tumor. Similarly to the 1847 col recognition, the reactivity by both T cell clones against 1872 col (Fig. 2A, B gray columns) was inhibited by anti-NKG2D mAb.

Taken together these data indicate that the anti-tumor reactivity can be either TCR or NKG2D driven, and that recognition of tumor target cells expressing NKG2D ligands can trigger its receptor leading to T cell activation. Our results suggest that the engagement by T cells of different receptors, depending on the complexity of the array of ligands available on the surface of tumor cells, may differentially contribute to stimulate the anti-tumor activity. Unfortunately, the limited life-span of these T lymphocytes prevented further investigations.

Figure 1.

 TCR- and NKG2D-mediated recognition of CRC lines by the CD8+ T cell clone M51. T cells (5×104) were incubated with target cells (5×104) in 96-well plate in 250-μl final volume of AIMV plus 2% HS. After 18 h the supernatants were collected and the content of IFN-γ evaluated by ELISA. Data represent pg/ml of IFN-γ * indicate the CRC lines used as target cells, 1869-B71 col (white bars), 1872 col (gray bars), 1847 col (black bars). These were pre-incubated for 48 h with 3,000 IU rhIFN-γ. Where indicated, tumor cells were pre-incubated at 37°C for 45 min with the anti-MHC class I (W6/32) or the anti-MICA mAb, while T cells were pre-incubated with anti-TCR and/or anti-NKG2D mAb (see Sect. 4). The data shown represent averages of three independent experiments with SD ≤15%; SD were calculated for all the samples, although some are too small and not visible in the figure. Statistical analysis of differences between the means of IFN-γ released by T cells have been performed by two-tailed t-test.

Figure 2.

 NKG2D-mediated anti-tumor activity by CD8+ clones M21 (A) and M23 (B). T cells (2×104) were incubated with target cells (2×104) in 96-well plate in a 250-μl final volume of AIMV 2% HS. After 18 h the supernatants were collected and the content of IFN-γ evaluated by ELISA. Data represent pg/ml of IFN-γ. * indicate the CRC lines used as targets, 1869-B71 col (white bar), 1872 col (gray bar), 1847 col (black bar). These were pre-incubated for 48 h with 3,000 IU rhIFN-γ. Where indicated, tumor cells were pre-incubated at 37°C for 45 min with mAb W6/32 or with the anti-MICA mAb, while T cells were pre-incubated with anti-NKG2D mAb. The data shown represent averages of three independent experiments with SD of ≤10%; SD were calculated for all the samples, although some are too small and not visible in the figure. Statistical analysis of differences between the means of IFN-γ released by T cells have been performed by two-tailed t-test.

2.3.2 NKG2D-independent tumor recognition by CD8+ T cell clones

CD8+ T cells exhibiting specific anti-tumor activity without NKG2D engagement were also isolated from PBMC of patient 1869. As shown in Fig. 3, the CD8+ T clone M83 exerted a class I-HLA-restricted recognition of the autologous CRC line1869-B7–1 col (white bars), with the IFN-γ secretion being almost completely inhibited in the presence of W6/32 mAb. In contrast, no inhibition occurred after incubation with the anti-NKG2D mAb and the reduction of cytokine released by W6/32 mAb plus anti-NKG2D was not significantly different from that achieved by W6/32 alone. The M83 clone also recognized, in HLA class I-restricted fashion, the allogeneic line 1870 col, sharing the HLA-A24 and -B35 molecules with the 1869 col, and this anti-tumor activity was not affected by incubation of T cells with anti-NKG2D mAb (black bars). Despite the fact that 1872 col cells share the HLA-A3 molecules and express NKG2D ligands, they were not recognized by M83 T cells (gray bars). The same occurred for the allogeneic lines 1847 col and SW480, although expressing NKG2D ligands. Furthermore, the failure of IFN-γ secretion by the M83 clone after incubation with the HLA class I- and NKG2D ligand-negative CRC LoVo line demonstrates the TAA-driven reactivity of these lymphocytes.

Taken together, the TCR-mediated anti-tumor activity of clone M83 and the predominant triggering of TCR in the recognition by M51 clone of the autologous tumor, suggest that in the presence of high-affinity interaction of TCR with MHC/peptide complexes, the involvement of NKG2D molecules is less relevant.

Figure 3.

 The engagement of TCR but not of NKG2D mediates the anti-tumor reactivity by the CD8+ T cell clone M83. T cells (2×104) were incubated with target cells (2×104) in 96-well plate in 250-μl final volume of AIMV + 2% HS. After 18 h supernatants were collected and the content of IFN-γ was evaluated by ELISA. Data of the y-axis represent pg/ml of IFN-γ. * indicate the CRC lines used as targets, 1869-B71 col (white bar), 1870 col (black bar), 1872 col (dark gray bar) 1847 col (black and white bar), and SW480 (clear gray bar). These were pre-incubated for 48 h with 3,000 IU rhIFN-γ. As indicated, tumor cells were pre-incubated at 37°C for 45 min with mAb W6/32, while T cells were pre-incubated with anti-NKG2D mAb. The data shown represent averages of three independent experiments with SD of ≤10%; SD were calculated for all the samples, although some are too small and not visible in the figure. Statistical analysis of differences between the means of IFN-γ released by T cells have been performed by two-tailed t-test.

2.4 NKG2D engagement in CD4+ T cell-mediated anti-tumor response

We also investigated whether the anti-tumor reactivity of the CD4+ T cell clones isolated from patient 1869 could be induced by engagement of the NKG2D molecules, in addition to the TCR triggering. We used the CD4+ T cell clone C111 isolated from 1869 PBMC and known to recognize a new HLA-DR-restricted CRC-associated antigen (COA-1) (Maccalli et al., manuscript in preparation). As shown in Fig. 4A, C111 lymphocytes recognized the autologous tumor cells expressing MHC class II molecules upon IFN-γ treatment (white bars) or after transduction with the transactivator factor CIITA (1869-CIITA col; black bars). Cytokine release was abolished after incubation of the target cells with the anti-HLA-DR mAb (L243). These T cells showed an HLA class II-restricted recognition of the allogeneic CRC line 1847 col (clear gray bars) and of melanoma line 1681 mel (dark gray bars) (see Fig. 4A), both sharing HLA-DRβ1 (DRβ1*1301 and DRβ1*0402, respectively) with the autologous 1869 col cells expressing the COA-1 antigen. The recognition of autologous tumor by clone C111 was weakly, but significantly, inhibited (16%, p<0.01) by treatment of the lymphocytes with the anti-NKG2D mAb, whereas, after stimulation with the 1847 col and the 1681 mel lines, IFN-γ secretion was inhibited after incubation of the effectors with the anti-NKG2D mAb (85% and 67%, respectively) or of target cells with the anti-MICA mAb (74% and 53% of inhibition, respectively) (Fig. 4A). These data indicate that the NKG2D has a role as costimulatory signal in CD4+ reactivity to NKG2D ligands highly expressed by tumor cells. However, in the case of the autologous 1869 col, the low level of its NKG2D ligands and the likely high affinity of TCR for MHC/peptide complexes limited an efficient engagement of the NKG2D receptor. Likewise, the evidence that allogeneic tumor lines, such as the 1847 col, pre-treated with IFN-γ to up-regulate MHC class II molecules, and thus expressing these molecules to the same level as the IFN-γ–treated autologous tumor (1869-B7–1 col) (data not shown), stimulated the C111 T cells more efficiently than the 1869 col, may be due to the enhancement of the T cell activation by the NKG2D-mediated costimulatory signal. The amount of cytokine released by the stimulation with the allogeneic CRC line 1847 col was similar to that observed with the CIITA transduced 1869 col (Fig. 4A), which expresses MHC class II molecules (data not shown) more efficiently, indicating that, in the absence of efficient NKG2D activation (such as in the case of the 1869 col), higher number of MHC/peptide complexes could be needed to achieve an efficient anti-tumor response. These results thus demonstrate, for the first time, a functional role of NKG2D for CD4+ T cell-mediated anti-tumor reactivity.

The CD4+/NKG2D T cell clone M26 was also generated from the mixed leukocyte/tumor cell cultures (MLTC) of the patient 1869. As shown in Fig. 4B, these T cells secreted IFN-γ (463 pg/ml) when cultured with autologous B7-1-transduced tumor cells, a reaction inhibited by the anti-HLA-DR mAb. The cytokine release was almost two times higher (819 pg/ml) when the CIITA-transduced autologous 1869 col was used as target cells, confirming that the level of the expression of the class II HLA can affect the anti-tumor reactivity by T cells. The M26 cells failed to recognized the allogeneic CRC line 1847 col, sharing the DRβ1*1301 molecules with the 1869 col (Fig. 4B). Specific HLA-DR-restricted reactivity of the M26 cells was observed against the allogeneic melanoma 1681 sharing the DRβ1*0402 molecules with the autologous tumor, suggesting that these T cells recognized an antigen in association with the DRβ1*0402 and expressed also by an allogeneic tumor of different histology. Since T cell clone M26 did not express the NKG2D receptor (data not shown), tumor cell recognition was achieved by TCR without contribution by the NKG2D receptor, as proven by the lack of the inhibition of tumor recognition by treatment of the effectors cells with the anti-NKG2D mAb or by incubation of the target cells with the anti-MICA mAb.

These results indicate that different anti-tumor CD4+ T cell clones can be isolated from one CRC patient that recognize heterogeneous target molecules, including MHC-presented epitopes and/or NKG2D ligands.

Figure 4.

 Anti-tumor reactivity of CD4+ T cell clones C111 and M26. T cells (5×104) were incubated with target cells (5×104) in 96-well plate in a 250-μl final volume of AIMV plus 2% HS. After 18 h supernatants were collected and the content of IFN-γ was evaluated by ELISA. Data of the y-axis represent pg/ml of IFN-γ. Data of T cell clones C111 and M26 are shown in (A) and (B), respectively. * indicate the tumor cell lines used as targets: 1869-B71 col (white bar), 1847 col (clear gray bar), 1681 mel (dark gray bar). Before being used as target, CRC cells were incubated for 48 h with 3,000 IU IFN-γ. The tumor line 1869-CIITA col (black bar) was transduced with the HLA transactivator factor to induce constitutive expression of MHC class II molecules. Where indicated, tumor cells were pre-incubated at 37°C for 45 min with mAb W6/32 or anti-MICA mAb, while T cells were pre-incubated with anti-NKG2D mAb. The data shown represent averages of two independent experiments with SD of ≤10%; SD were calculated for all the samples, although some are too small and not visible in the figure. Statistical analysis of differences between means of IFN-γ released by T cells have been performed by two-tailed t-test.

3 Discussion

We have investigated whether, in addition to TCR, T cell-mediated anti-tumor reactivity may be induced by the engagement of NKG2D. By examining the expression of NKG2D ligands (MICA/B and ULBP) by tumor cell lines, including freshly established CRC lines, we found an heterogeneous expression of MICA/B and ULBP molecules. ULBP2 molecules were commonly expressed, although not homogeneously, by the CRC lines analyzed (five out of seven) and, moreover, by tumor lines negative for MICA. Only two lines (1870 col and 1847 col) expressed high level of ULBP3. In addition, neither MICB not ULBP1 (except for a weak expression of ULBP-1 by 1870 col and SW480) were detected on the surface of the CRC lines, suggesting that these molecules may be differentially regulated or that defective expression may occur in tumor cells. Polymorphic modifications of the MICA gene resulting in its lack of surface expression have been described 17. This analysis led us to conclude that the NKG2D ligands are heterogeneously expressed by CRC lines, and that MICA-negative tumors can express other NKG2D ligands, such as ULBP. Detailed studies are needed to investigate the regulation of NKG2D ligands in tumor cells and to assess whether a post-translational regulation of these molecules may occur, as suggested by the HCMV model 18.

We generated both CD8+ and CD4+ T cell clones from patient 1869 that displayed an array of anti-tumor specificities. In fact, four CD8+ T cell clones recognized the autologous tumor in class I HLA-restricted fashion. However, M51 T lymphocytes also reacted against the allogeneic 1872 col and 1847 col CRC lines independently from HLA/peptide complexes recognition. Furthermore, NKG2D mediated the autologous tumor recognition by this clone, although the reactivity directed to autologous tumor cells was less affected by blocking with the anti-NKG2D mAb than by blocking with anti-TCR or anti-HLA class I mAb. In contrast, the allogeneic reactivity was dependent on NKG2D stimulation, since it correlated with significant levels of expression of MICA and ULBP. A similar pattern of reactivity was observed for other CD8+ clones (M21 and M23) that released IFN-γ after incubation with either the autologous or allogeneic CRC lines. This indicates that for these effectors either TCR or NKG2D mediate the recognition of autologous 1869 col, whereas the allogeneic tumor reactivity, in some instances, can be dependent even on the triggering of NKG2D alone. With the autologous tumor as target cells, the TCR-mediated signal was not predominant (see Fig. 2), probably depending on a low affinity of the TCR for the HLA/peptide complexes that can in turns lead to the involvement of NKG2D to elicit a significant anti-tumor response. The specific tumor reactivity of these T cell clones was demonstrated by the lack of recognition of the HLA class I-negative LoVo cell line and by the absence of reactivity against normal B and EBV-transformed B cells.

These observations appear to be at variance with previous data on the anti-viral reactivity of NKG2D+ T cells 13. In that model, the NKG2D has been described as a costimulatory signal for the activation of the immune effectors cells that needed the concomitant engagement of both TCR and NK activatory receptors. However, the NKG2D-dependent killing of tumor lines by NK cells has been documented 10. Indeed, our data suggest that. when CRC cells expressed simultaneously high level of different NKG2D ligands, or when the affinity of TCR for MHC/peptides complexes is low, the triggering of NKG2D alone can lead to the anti-tumor immune response. Of note, when NKG2D was the only engaged receptor, the cytokine secretion was lower than after costimulation of TCR, suggesting that more efficient anti-tumor reactivity is achieved when TCR is engaged and its signal could be strengthened by co-engagement of NKG2D. Support for the complexity of the signals delivered by NKG2D was given in the results of a previous study 14 showing that MICA can deliver both the TCR and the NKG2D-dependent costimulatory signal in γ δ T cells, and providing further evidence that the engagement of TCR and NKG2D might depend on the array of ligands available on the target cells and on the studied model. In addition, ULBP expression by NK cell-resistant target cells stimulated cytotoxic activity by NK cells, despite the triggering of other activatory receptors 6, 7. However, NKG2D ligands may represent target molecules for T cell-mediated immune response when HLA-mediated stimulation is impaired, as occurs when HLA molecules are down-regulated during viral infection or neoplastic transformation 6, 19, 20.

On the other hand, the usage of NKG2D receptor to activate a specific anti-CRC tumor response by CD8+ T cells is not a general phenomenon. The T cell clone M83, isolated from the same 1869 PBMC culture, exerted exclusively HLA class I-mediated reactivity directed to the autologous CRC cells and to the allogeneic, HLA-A24 sharing, 1870 col line. Our data thus indicate that CD8+ T cell clones isolated from the peripheral blood of a CRC patient can display anti-tumor activity, with heterogeneous engagement of different activatory receptors depending on the level of expression of their ligands and on the affinity with the specific TAA expressed by the CRC cells (Table 3).

Moreover, the analysis of the anti-colon cancer CD4+ T cells demonstrated that a more efficient anti-tumor activity can be achieved by the cooperation of both TCR and NKG2D receptors. In fact, the autologous tumor reactivity of the T cell clone C111 was mainly mediated by TCR engagement, as shown by the weak blocking activity of the anti-NKG2D mAb, whereas the allogeneic reactivity was significantly reduced by incubation of effectors with anti-NKG2D mAb. Therefore, in this case allogeneic tumor reactivity was both TCR and NKG2D dependent, confirming that the efficient expression of the NKG2D ligands is the limiting factor for NKG2D triggering. For C111 T cells, NKG2D can deliver a costimulatory signal together with the requirement of the TCR-mediated stimulation. Thus, also for CD4+ T cells the heterogeneity of the receptors that can deliver the activatory signals lead to different types of tumor reactivity. In addition, we have described the first example of CD4+ T cell clones that express NKG2D and thefunctional role of this receptor in anti-tumor activity.

We have shown that the NKG2D-mediated signal can affect the pattern of specificity of the tumor-reactive T cells, and that the anti-tumor response can be strengthened by the involvement of multiple receptors that recognized specific ligands on target cells. Unfortunately, the limited life-span of these T cell clones prevented a more detailed investigation of this aspect of the immune response to tumors.

Recently, it has been shown for human melanoma that one of the NKG2D ligands (MICA) can induced down-modulation of NKG2D on T cells and in tumor-infiltrating T lymphocytes 21. Furthermore, this phenomenon is associated with the presence of the circulating tumor-derived soluble MICA, suggesting that this mechanism might cooperate with the immune evasion of tumors. Our data indicate that the heterogeneous expression of different NKG2D ligands by tumor cells may limit the T cell impairment, since down-modulation of all the NKG2D ligands is unlikely to occur, similarly to the rarely occurring loss of the complete HLA haplotype in tumors 22. Since the down-modulation of NKG2D ligands has been described in metastatic tumors, the same analysis performed in tumors at early stage of the disease will help to determine whether NKG2D+ T cells represent relevant anti-tumor effectors during the early steps of tumor development.

Table 3. Summary of the activatory receptors engaged by the CD8+ T cell clones in the recognition of CRC lines.
  1. a) HLA class I shared with autologous tumor.

  2. b) The expression of NKG2D ligands (MICA/B and ULBP-1, -2 and -3) has been indicated on the basis of the data reported in Table 1 and the number of + represents theamount of different NKG2D ligands expressed by tumor cells.

  3. c) Activatory receptors that mediate the anti-tumor activity by T cells (see also Fig. 1-3); –: no reactivity.

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4 Materials and methods

4.1 Establishment and characterization of CRC lines

CRC lines (1869 col, 1870 col, 1872 col, and 1847 col) have been established in vitro from tumor specimens of CRC patients admitted at the Surgery Branch, National Cancer Institute (National Institute of Health, Bethesda, MD) (Maccalli et al., manuscript in preparation). Briefly, the tumor cells were cultured at the early passages on collagen-coated six-well plates (Biocoated plates, Becton Dickinson, Franklin Lakes, NJ) with the ACL-4 medium 23 (InVitrogen, Carlsbard, CA) plus 10 ng/ml epidermal growth factor, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 50 μg/ml gentamicin, 50 ng/ml amphotericin-B (MEGM Single Quotes, Clonetics, Walkersville, MD) and 10% fetal bovine serum (FBS) (BioWhittaker, Walkersville, MD) at 37°C in 5% CO2. The established tumor lines were then cultured with RPMI 10% FBS. These cell lines have been characterized by immunofluorescence and FACS analysis (FACScan, Becton Dickinson, Franklin Lakes, NJ) for the expression of HLA classes I and II by the mAb W6/32 and L243 (Becton Dickinson, CA), respectively (data not shown).

4.2 Other cell lines

The melanoma line 1681mel and the EBV-transformed B cell line 1869 were generated at the Surgery Branch and were cultured in RPMI plus 10% FBS. The PBMC-derived freshly generated B cell lines 1847 CD40L-B, 1869 CD40L-B and 1872 CD40L-B were cultured, as previously described 24, in Iscove's medium (GIBCO) plus 10% human serum (HS) in the presence of 100 IU/ml CD40L(Immunex, Seattle, WA) and 100 IU/ml recombinant human (rh) IL-4 (PharMingen, San Diego, CA). The colon cancer lines SW1463, SW480, WIDR and LoVo were obtained from ATCC (CCL-234, CCL-228, CCL-218 and CCL-229, American Tissue Culture Collection, Manassas, VA) and were cultured in RPMI plus 10% FBS. The MHC class I and class II typing of the PBMC and tumor lines used in this study was performed by single-stranded oligonucleotide probe-PCR typing. The 1869 col line was transduced with a retroviral vector encoding the costimulatory molecule B7-1 25 and used to stimulate autologous PBMC and as target cells in cytokine secretion assay. To induced a stable and efficient expression of MHC class II molecules, the CRC line 1869 col was also transduced with a retroviral vector encoding the CIITA transactivator factor 26 (data not shown).

4.3 Flow cytometry analysis

The expression of MICA, MICB and ULBP molecules by the tumor lines was determined by FACS analysis using the following mAb: anti-MICA BAM195 supernatant generated by one of us (D.P.) and purified anti-MICA M673, anti-MICB M362, anti-ULBP1 M295, anti-ULBP2 M310 and anti-ULBP3 M551 (all from Immunex). The phenotype analysis of the 1869-B71 or -CIITA col lines showed that the expression of surface markers was not affected by retroviral transduction of the cells (data not shown).

The expression of NKG2D by anti-tumor T lymphocytes was similarly evaluated using BAT221 mAb 8 supernatant and the purified M580 mAb (Immunex). The PE-conjugated goat anti-mouse immunoglobulins (DAKO, Denmark) were used for fluorochrome staining of the mAb used. The phenotype characterization of the specific T cell clones isolated from PBMC of a CRC patient was performed by immunofluorescence and cytofluorimetric analysis with the PE- or FITC-conjugated anti-CD3, -CD4, -CD8, -CD16, or -CD56 mAb (Becton Dickinson). The cytofluorimetric analysis was performed usinga FACScan instrument (Becton Dickinson).

4.4 Isolation of anti-tumor T cells and cytokine release assay

T lymphocytes were isolated from PBMC of the CRC patient 1869 (HLA-A3, 24; -B35, 38; -CW4, 12; -DRβ1*0404,1301). The T lymphocytes were stimulated in vitro with autologous irradiated (150 Gy) B7–1 gene-transfected tumor cells (tumor cells:lymphocytes ratio 1:5), and rhIL-2 (300 IU/ml), in RPMI (BioWhittaker) plus 10% HS. The cultures were re-stimulated weekly for 3 weeks. The tumor cells used for T lymphocytes stimulation were cultured for at least 10 days in RPMI plus 10% HS. The cell clones were generated from the established bulk culture by limiting dilution in the presence of allogeneic irradiated (50 Gy) PBMC (1×105 cells/well), autologous irradiated (150 Gy) tumor cells (1×103 cells/well) and 1 μg/ml PHA (Sigma) and were cultured with RPMI plus 10% HS. These cultures were weekly stimulated and, after 2 weeks, T cell growth was evaluated. The reactivity of the generated T cell clones against autologous and allogeneic CRClines was examined by IFN-γ secretion assay. T cells were incubated (1.5×104–5×104 cells/well) in flat-bottom 96-well plates in the presence of tumor cells (1.5×104–5×104 cells/well). After 18 h of incubation at 37°C and 5% CO2, the supernatants were collected and the IFN-γ released by T cells was evaluated by ELISA (anti-IFN-γ coating mAb, M700-A-E, and anti-IFN-γ biotinylated mAb M701-B, Endogen, Rockford, IL). The specificity of the T lymphocytes was assessed by inhibition of IFN-γ release after pre-incubaton of the target cells with 10 μg/ml of either anti-HLA class I mAb W6/32, or anti-HLA-DR class II mAb L243, or with 50 μl/well anti-MICA mAb BAM195. The inhibition of IFN-γ release was also evaluated following the pre-incubation of T lymphocytes with 50 μl/well anti-NKG2D mAb BAT221 or with 10 μg/ml anti-TCR mAb (TCR1043, Endogen). Statistical analysis of differences between means for cytokine-release assay was done using two-tailed t-test.

Acknowledgements

We thank Dr. David Cosman, Department of Immunobiology and Molecular Biology, Amgen Corporation (Seattle, WA) for providing mAb directed to NKG2D and its ligands. This work was partially supported by Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan, Italy) and by European Community (QLK3 1999 00064). C. Maccalli was supported by a fellowship of the Istituto Nazionale Tumori from "Successione Bertaccini".

Footnotes

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