p53 potentiation of tumor cell susceptibility to CTL involves Fas and mitochondrial pathways

In this study, we have investigated the mechanisms used by wild-type p53 (wtp53) to potentiate tumor cell susceptibility to CTL-mediated cell death. We report that wtp53 restoration in a human lung carcinoma cell line Institut Gustave Roussy (IGR)-Heu, displaying a mutated p53, resulted in up-regulation of Fas/CD95 receptor expression associated with an increase of tumor cell sensitivity to the autologous CTL clone, Heu127. However, when IGR-Heu cells were transfected with Fas cDNA, no potentiation to Heu127-mediated lysis was observed, indicating that induction of CD95 is not sufficient to sensitize target cells to CTL killing. Importantly, our data indicate that the effect of wtp53 on the Fas-mediated pathway involves a degradation of short cellular FLICE inhibitory protein resulting in subsequent caspase 8 activation. Furthermore, we demonstrate that wtp53 restoration also resulted in CTL-induced Bid translocation into mitochondria and a subsequent mitochondrial membrane permeabilization leading to cytochrome c release. These results indicate that tumor cell killing by autologous CTL can be enhanced by targeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machinery regulation.     

Treatment of human colon carcinoma cell lines with anti-neoplastic agents enhances their lytic sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes

Certain anti-neoplastic agents at subtoxic doses may exert immunomodulatory effects, which alter the expression of specific tumor cell surface molecules. We reasoned that potential increases in tumor cell surface markers, such as those important for facilitating effector-target contact, as well as triggering cell death pathways, might then improve antigen (Ag)-specific T-cell-mediated tumor cytolysis. Here, in a human colon carcinoma cell model in vitro, we examined whether the anti-neoplastic agents 5-fluorouracil (5-FU), CPT-11 or cisplatin (CDDP) could upregulate the expression of specific tumor cell surface markers, which may then enhance productive lytic interactions between CD8+ CTL and Ag-bearing tumor cells. Based on our earlier studies, IFN-gamma treatment was included as a control for sensitization to CTL-mediated lysis. Pretreatment of the SW480 primary colon carcinoma cell line with IFN-gamma, 5-FU, CPT-11 or CDDP enhanced ICAM-1 and Fas expression, resulting in Ag-specific CTL-mediated lysis involving Fas-dependent and -independent mechanisms. In contrast, pretreatment of the SW620 metastatic isolate, derived from the same patient, with IFN-gamma, CPT-11 or CDDP, but not 5-FU, enhanced ICAM-1 expression, resulting in Ag-specific CTL-mediated lysis via Fas-independent mechanisms only. Flow cytometric-based assays were then developed to measure the effects of drug treatment on caspase signaling and apoptosis incurred by tumor targets after interaction with CTL. We found that the lytic enhancement caused by drug treatment of SW480 or SW620 targets was accompanied by an increase in caspase-3-like protease activity. A peptide-based caspase inhibitor abrogated CTL-mediated apoptosis, suggesting that "chemomodulation" involved regulation of the caspase pathway. These results revealed for the first time an important role for components of the caspase pathway, such as caspase-3-like proteases, in the sensitization of human colon carcinoma cells by anti-neoplastic agents to Ag-specific CTL. Thus, certain anti-neoplastic agents may display unique immunoregulatory properties that facilitate human colon carcinoma death by engaging the lytic capacity of Ag-specific CTL, which may have implications for chemoimmunotherapy strategies.     

Tumor-infiltrating CD4+ T lymphocytes express APO2 ligand (APO2L)/TRAIL upon specific stimulation with autologous lung carcinoma cells: role of IFN-alpha on APO2L/TRAIL expression and -mediated cytotoxicity

In the present report, we have investigated TRAIL/APO2 ligand (APO2L) expression, regulation, and function in human lung carcinoma tumor-infiltrating lymphocytes. Using a panel of non-small cell lung carcinoma cell lines, we first showed that most of them expressed TRAIL-R1/DR4, TRAIL-R2/DR5, but not TRAIL-R3/DcR1 and TRAIL-R4/DcR2, and were susceptible to APO2L/TRAIL-induced cell death. Two APO2L/TRAIL-sensitive tumor cell lines (MHC class I(+)/II(+) or I(+)/II(-)) were selected and specific CD4(+) HLA-DR- or CD8(+) HLA-A2-restricted CTL clones were respectively isolated from autologous tumor-infiltrating lymphocytes. Interestingly, although the established T cell clones did not constitutively express detectable levels of APO2L/TRAIL, engagement of their TCR via activation with specific tumor cells selectively induced profound APO2L/TRAIL expression on the CD4(+), but not on the CD8(+), CTL clones. Furthermore, as opposed to the CD8(+) CTL clone which mainly used granule exocytosis pathway, the CD4(+) CTL clone lysed the specific target via both perforin/granzymes and APO2L/TRAIL-mediated mechanisms. The latter cytotoxicity correlated with APO2L/TRAIL expression and was significantly enhanced in the presence of IFN-alpha. More interestingly, in vivo studies performed in SCID/nonobese diabetic mice transplanted with autologous tumor and transferred with the specific CD4(+) CTL clone in combination with IFN-alpha resulted in an important APO2L/TRAIL-mediated tumor growth inhibition, which was prohibited by soluble TRAIL-R2. Our findings suggest that APO2L/TRAIL, specifically induced by autologous tumor and up-regulated by IFN-alpha, may be a key mediator of tumor-specific CD4(+) CTL-mediated cell death and point to a potent role of this T cell subset in tumor growth control.     

Resistance of tumor cells to cytolytic T lymphocytes involves Rho-GTPases and focal adhesion kinase activation

Tumor cells evade adaptive immunity by a variety of mechanisms, including selection of variants that are resistant to specific cytotoxic T lymphocyte (CTL) pressure. Recently, we have reported that the reorganization of the actin cytoskeleton can be used by tumor cells as a strategy to promote their resistance to CTL-mediated lysis. In this study, we further examined the functional features of a CTL-resistant tumor variant and investigated the relationship between cytoskeleton alteration, the acquisition of tumor resistance to CTL-induced cell death, Rho-GTPases, and focal adhesion kinase (FAK) pathways. Our data indicate that although the resistant cells do not display an increased migratory potential, an alteration of adhesion to the extracellular matrix was observed. When Rho-GTPases were activated in cells by the bacterial CNF1 (cytotoxic necrotizing factor 1), striking changes in the cell morphology, including actin cytoskeleton, focal adhesions, and membrane extensions, were observed. More importantly, such activation also resulted in a significant attenuation of resistance to CTL-induced cell death. Furthermore, we demonstrate that FAK signaling pathways were constitutively defective in the resistant cells. Silencing of FAK in the sensitive target cells resulted in the inhibition of immune synapse formation with specific CTLs and their subsequent lysis. Expression of the FAK mutant (Y397F) resulted in an inhibition of IGR-Heu cell adhesion and of their susceptibility to specific lysis. These results suggest that FAK activation plays a role in the control of tumor cell susceptibility to CTL-mediated lysis.     

Adoptive immunotherapy of syngeneic murine leukemia is enhanced by the combination of recombinant IFN-gamma and a tumor-specific cytotoxic T cell clone

Mice with an established syngeneic T cell tumor (RBL5) received short term adoptive chemoimmunotherapy with CTL clone 1.B6 and murine rIFN-gamma. In comparison with treatment with either agent alone, the combination of 1.B6 and rIFN-gamma was associated with a dramatic increase in long term survival. No direct effects of rIFN-gamma on tumor cell proliferation, MHC Ag expression, or susceptibility to CTL-mediated lysis could be demonstrated to explain the prolongation of survival. However, rIFN-gamma induced a distinct increase in broad-spectrum cytolytic capacity of peritoneal exudate cells and further increased class II MHC expression on peritoneal macrophages. The explanation for enhanced adoptive chemoimmunotherapy after combined short term administration of a CTL clone and rIFN-gamma is uncertain. Potential mechanisms include direct tumor lysis by activated cells, indirect tumor lysis via sensitization to other lymphokines or monokines, improved Ag-specific activation of transferred CTL clones, and/or more effective development of de novo host anti-tumor immunity.     

Characterization of CD8+ cytotoxic T lymphocyte/tumor cell interactions reflecting recognition of an endogenously expressed murine wild-type p53 determinant

p53 mutations are frequently found in human cancers and are often associated with the overexpression of wild-type (WT) protein or peptide sequences, supporting the notion that WT p53 epitopes may serve as potential targets for tumor immunotherapy. We have developed a cytotoxic T lymphocyte (CTL)/p53 tumor-associated antigen (TAA) model, based on immune recognition of a WT p53 determinant. WT p53-peptide-specific, major histocompatibility complex (MHC) classI-restricted CTL were produced from immunocompetent C57BL/6 (H-2b) mice after immunization with a previously defined WT p53 peptide (p53(232-240)) Epitope-specific CTL were then employed to identify syngeneic tumor cell populations expressing that antigenic determinant. Two syngeneic tumor cell lines, MC38 colon carcinoma and MC57G fibrosarcoma, were demonstrated to express the endogenous WT p53(232-240) determinant naturally, as defined by CD8 + CTL recognition. Cold-target inhibition assays confirmed that CTL-mediated lysis was due to immune recognition of the p53(232-240) peptide epitope. The p53(232-240)-specific CTL line did not lyse syngeneic normal cells (i.e., mitogen-activated splenocytes) in the absence of exogenous peptide, suggesting that the WT-p53-specific CTL could distinguish between tumor cells expressing self-TAA and normal host cells. We have demonstrated, for the first time, that the adoptive transfer of WT-p53-specific CTL to mice with established pulmonary metastasis resulted in antitumor activity in vivo. The ability to generate MHC-class-I-restricted CD8- CTL lines specific for a non-mutated p53 determinant from normal, immunocompetent mice, which display antitumor activity both in vitro and in vivo (by adoptive transfer), may have implications for the immunotherapy of certain p53-expressing malignancies.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

Influence of interferon γ on modulation of Fas expression by human colon carcinoma cells and their subsequent sensitivity to antigen-specific CD8+ cytotoxic T lymphocyte attack

The inability of certain neoplastic populations to undergo Fas-mediated death by immune effector mechanisms may confer a selective survival advantage, which may contribute to tumor escape. In this study, we examined the role of Fas-mediated lysis in a human-antigen (Ag)-specific cytotoxic T lymphocyte (CTL)/colon carcinoma cell model, and the regulation of the lytic phenotype by interferon γ (IFNγ). Previously, we have identified mutated ras peptides reflecting the valine-for-glycine substitution at position 12 as unique HLA-A2-restricted, CD8⁺ CTL neo-epitopes. Peptide-specific CTL, established from both normal and carcinoma-bearing individuals, lysed in vitro a HLA-A2⁺ primary colon adenocarcinoma cell line, SW480, harboring the naturally occurring ras mutation. Pretreatment of SW480 cells with IFNγ was necessary to promote efficient Ag-specific CTL killing, although the mechanisms by which IFNγ influenced the lytic outcome remains to be elucidated. Here, we show, by phenotypic analysis of SW480 cells, a significant up-regulation of HLA-A2, ICAM-1 and Fas molecules after IFNγ pretreatment, which paralleled their sensitivity to lysis with anti-Fas stimuli. Moreover, nearly half of the lytic response to IFNγ-treated SW480 cells was inhibited by neutralizing anti-Fas or anti-Fasligand (FasL) mAb, revealing for the first time an important functional role for Fas/FasL interactions in carcinoma cell killing by human Ag-specific CTL. mAb against HLA-A2, ICAM-1, the αβ T cell receptor (TCR) and Fas molecules inhibited lysis; however, if these CTL were preactivated to express functional FasL and then used as effectors, only anti-Fas mAb efficiently blocked lysis. IFNγ also increased pro-caspase-3 protein expression and its subsequent activation in SW480 cells following Ag-specific CTL attack. Peptide-based caspase inhibitors blocked both caspase-3 activation and CTL-mediated lysis. Overall, these data suggested that IFNγ (a) facilitated both Ag-dependent and Ag-independent events as a prerequisite for efficient CTL/target interactions, FasL up-regulation and triggering of Fas-dependent, as well as Fas-independent lysis (perforin); and (b) enhanced or restored a Fas-sensitive phenotype in SW480 cells, reflecting modulation of cell-surface and intracellular elements of the Fas pathway. Thus, IFNγ may play an important role in the regulation of a human neoplastic cell death phenotype, which may have implications for our understanding of the processes of both tumor evasion and tumor regression following Ag-specific CTL attack. Received: 20 December 1999 / Accepted: 1 February 2000     

Granzyme B-induced cell death involves induction of p53 tumor suppressor gene and its activation in tumor target cells

In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-alpha-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-alpha resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.     

Differential role of Fas/Fas ligand interactions in cytolysis of primary and metastatic colon carcinoma cell lines by human antigen-specific CD8+ CTL

We have previously identified mutated ras peptides reflecting the glycine to valine substitution at position 12 as HLA-A2-restricted, CD8+ CTL neo-epitopes. CTL lines produced against these peptide epitopes lysed the HLA-A2+ Ag-bearing SW480 primary colon adenocarcinoma cell line, although IFN-gamma treatment of the targets was necessary to achieve efficient cytotoxicity. Here, we compared the lytic phenotype of the SW480 cell line to its metastatic derivative, SW620, as an in vitro paradigm to further characterize the nature of a HLA class I-restricted, Ag-specific CTL response against neoplastic cell lines of primary and metastatic origin. Although both colon carcinoma cell lines were lysed by these Ag-specific CTL following IFN-gamma pretreatment, the mechanisms of lysis were distinct, which reflected differential levels of sensitivity to the Fas pathway. Whereas IFN-gamma pretreatment rendered SW480 cells sensitive to both Fas-dependent and -independent (perforin) pathways, SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. Moreover, pretreatment of SW480 cells with the anti-colon cancer agent, 5-fluorouracil (5-FU), led to enhanced Fas and ICAM-1 expression and triggered Ag-specific CTL-mediated lysis via Fas- and perforin-based pathways. In contrast, these phenotypic and functional responses were not observed with SW620 cells. Overall, these data suggested that 1) IFN-gamma and 5-FU may enhance the lytic sensitivity of responsive colon carcinoma cells to immune effector mechanisms, including Fas-induced lysis; 2) the malignant phenotype may associate with resistance to Fas-mediated lysis in response to Ag-specific T cell attack; and 3) if Ag-specific CTL possess diverse lytic capabilities, this may overcome, to some extent, the potential "escape" of Fas-resistant carcinoma cells.     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

Tumor-specific CTL kill murine renal cancer cells using both perforin and Fas ligand-mediated lysis in vitro, but cause tumor regression in vivo in the absence of perforin

Kidney cancer is a devastating disease; however, biological therapies have achieved some limited success. The murine renal cancer Renca has been used as a model for developing new preclinical approaches to the treatment of renal cell carcinoma. Successful cytokine-based approaches require CD8(+) T cells, but the exact mechanisms by which T cells mediate therapeutic benefit have not been completely identified. After successful biological therapy of Renca in BALB/c mice, we generated CTLs in vitro using mixed lymphocyte tumor cultures. These CTL mediated tumor-specific H-2K(d)-restricted lysis and production of IFN-gamma, TNF-alpha, and Fas ligand (FasL) in response to Renca. CTL used both granule- and FasL-mediated mechanisms to lyse Renca, although granule-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-gamma and TNF-alpha increased the sensitivity of Renca cells to CTL lysis by both granule- and FasL-mediated death pathways. Adoptive transfer of these anti-Renca CTL into tumor-bearing mice cured most mice of established experimental pulmonary metastases, and successfully treated mice were immune to tumor rechallenge. Interestingly, we were able to establish Renca-specific CTL from mice gene targeted for perforin (pfp(-/-)) mice. Although these pfp(-/-) CTL showed reduced cytotoxic activity against Renca, their IFN-gamma production in the presence of Renca targets was equivalent to that of wild-type CTL, and adoptive transfer of pfp(-/-) CTL was as efficient as wild-type CTL in causing regression of established Renca pulmonary metastases. Therefore, although granule-mediated killing is of paramount importance for CTL-mediated lysis in vitro, some major in vivo effector mechanisms clearly are independent of perforin.     

Membrane structures involved in auto-tumor recognition

Tumor patients' blood lymphocytes have the capacity to recognize autologous tumor cells in vitro. A consequence of this recognition is the proliferation of small-size, high-density, resting T cells. Both helper (CD4+) and cytotoxic/suppressor (CD8+) T lymphocytes proliferate in the mixed lymphocyte-tumor cell cultures. In contrast to the autologous mixed lymphocyte cultures, both the auto-erythrocyte rosetting and non-rosetting (AE+ and AE-) T cells participate in the auto-tumor response. In contrast to stimulation by virus-infected or hapten-modified cells, DR antigen expression is not essential for stimulation by autologous tumor cells. In a proportion of cancer patients, blood lymphocytes have the capacity to lyse the patients' own tumor cells in vitro. There are two populations of lymphocytes with auto-tumor cytotoxic function. The first is characterized by low buoyant density and by non-adaptive cytotoxicity. In contrast to the recognition of hapten-modified or virus-infected target cells by the CTL, recognition of autologous tumor cells by the cytotoxic LD cells occurs even when the MHC class I antigens are blocked by mAb. The CD3 complex is also not involved in LD-mediated lysis. The other population with auto-tumor cytotoxic function comprises high-density, resting T cells. Recognition of autologous tumor cells by cytotoxic HD lymphocytes shares the characteristics of CTLs, i.e., their function is abrogated by pretreatment of the effectors with mAbs directed to the T3 receptor complex and by preincubation of the targets with mAb to the MHC class I antigens. Cytotoxicity of HD cells is restricted to the autologous tumor cells. This selectivity and the characteristics shared with CTL suggest that the auto-tumor reactivity of HD lymphocytes reflects an immune response against the autologous tumor.     

Simultaneous cellular and humoral immune response against mutated p53 in a patient with lung cancer

We recently identified several Ags recognized by tumor-infiltrating B lymphocyte-derived Ab using SCID mice and a xenografted non-small cell lung cancer system. One of these identified Ags was mutated p53 with a point mutation resulting in the alteration of codon 158 from Arg to Leu. The aim of this study was to ascertain whether cellular immunity against mutated p53 exists in the same patient together with humoral immunity. Two different nona peptides (mutated p53(150) and p53(155) peptides), including a mutated amino acid derived from p53, were synthesized according to the binding motif of HLA class I of the established cancer cell line A904L from the patient. Mediastinal lymph node lymphocytes of the patient were stimulated weekly with the peptides. The mutated p53(155) peptide-stimulated lymphocytes showed specific cytotoxicity against both autologous EBV-transformed B cells pulsed with mutated p53(155) peptide and A904L. The mutated p53(155) peptide-specific CTL clone in an HLA-Cw*0702 restriction was established and analyzed for its TCR usage. Clonotypic PCR using CDR3-specific primers was applied to the tumor tissue containing the tumor-infiltrating lymphocytes. The specific amplification of PCR was found in the tumor tissue. These results demonstrated that not only B lymphocytes producing specific Ab against the p53 protein, but also CTL against mutated p53, expressed in autologous lung cancer cells exist in the tumor tissue. This approach may allow us to better understand the mechanisms of T and B cell immunity against the same tumor Ag in cancer patients.     

A Common Role for Target Cell Histocompatibility Antigens in Both Nonspecific and Specific T Lymphocyte-Mediated Cytolysis

We have investigated the role of target cell major histocompatibility complex antigens (MHC-Ag) in nonspecific lectin-dependent lymphocyte-mediated cytolysis (LDCC). In contrast to previous reports, we provide evidence that in LDCC the lectin Concanavalin A (Con A) does not mediate lysis by simply bridging cytotoxic T lymphocytes (CTL) and targets via cell surface sugars or by activating the lytic function of CTLs attached to targets via the lectin. Lysis occurs when target cells are pretreated with lectin, but not when CTL are pretreated. Moreover, when CTL populations are used as both aggressors and targets, and only one is pretreated with lectin, lysis occurs only in the direction of the pretreated CTL target. We have observed that in LDCC, as in specific CTL-mediated killing, target recognition proceeds through interaction of CTL receptors (distinct from sugar moieties) and target cell surface determinants perhaps modified by, but distinct from, the lectin itself. We present evidence that the target determinants recognized in LDCC are MHC-Ag: 1) Cells that display reduced amounts of MHC-Ag are poor targets in LDCC; 2) removal of MHC-Ag by papain renders targets refractory to LDCC, however susceptibility is regained upon regeneration of MHC-Ag; and 3) antisera to target cell MHC-Ag block LDCC. The latter finding is also observed in oxidation-dependent CTL-mediated cytotoxicity. Involvement of MHC proteins in both specific and nonspecific CTL-mediated lysis reconciles an apparent fundamental distinction between these two processes and suggests a possible role for MHC proteins in a postrecognition step(s) leading to lysis.     

Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.     

Fas-ligand-mediated lysis of erbB-2-expressing tumour cells by redirected cytotoxic T lymphocytes

A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor γ chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-γ clones, and lysis of erbB-2⁺ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-γ, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL. Received: 23 July 1998 / Accepted: 5 October 1998     

CTL adoptive immunotherapy concurrently mediates tumor regression and tumor escape

Tumor escape and recurrence are major impediments for successful immunotherapy. It is well-documented that the emergence of Ag-loss variants, as well as regulatory mechanisms suppressing T cell function, have been linked to inadequate antitumor activity. However, little is known regarding the role of Fas-mediated cytotoxicity by tumor-specific CD8(+) CTL in causing immune evasion of Fas resistant variants during adoptive immunotherapy. In this study, we made use of an adoptive transfer model of experimental lung metastasis using tumor-specific CTL as a relevant immune-based selective pressure, and wherein the Fas ligand pathway was involved in the antitumor response. Surviving tumor cells were recovered and examined for alterations in antigenic, functional, and biologic properties. We showed that diminished susceptibility to Fas-mediated cytotoxicity in vivo was an important determinant of tumor escape following CTL-based immunotherapy. Tumor escape variants (TEV) recovered from the lungs of CTL-treated mice exhibited more aggressive behavior in vivo. However, these TEV retained relevant MHC class I and tumor Ag expression and sensitivity to CTL via the perforin pathway but reduced susceptibility to Fas-mediated lysis. Moreover, TEV were significantly less responsive to eradication by CTL adoptive immunotherapy paradigms as a consequence of increased Fas resistance. Overall, we identified that Fas(low)-TEV emerged as a direct consequence of CTL-tumor interactions in vivo, and that such an altered neoplastic Fas phenotype compromised immunotherapy efficacy. Together, these findings may have important implications for both tumor progression and the design of immunotherapeutic interventions to confront these selective pressures or escape mechanisms.     

Hypoxia-induced autophagy: a new player in cancer immunotherapy?

A major challenge in formulating an effective immunotherapy is to overcome the mechanisms of tumor escape from immunosurveillance. We showed that hypoxia-induced autophagy impairs cytotoxic T-lymphocyte (CTL)-mediated tumor cell lysis by regulating phospho-STAT3 in target cells. Autophagy inhibition in hypoxic cells decreases phospho-STAT3 and restores CTL-mediated tumor cell killing by a mechanism involving the ubiquitin proteasome system and SQSTM1/p62. Simultaneously boosting the CTL-response, using a TRP-peptide vaccination strategy, and targeting autophagy in hypoxic tumors, improves the efficacy of cancer vaccines and promotes tumor regression in vivo. Overall, in addition to its immunosuppressive effect, the hypoxic microenvironment also contributes to immunoresistance and can be detrimental to antitumor effector cell functions.