Research Article

Immunology and Cell Biology (1998) 76, 350–356; doi:10.1046/j.1440-1711.1998.00758.x

Drug resistance results in alterations in expression of immune recognition molecules and failure to express Fas (CD95)

A Bhushan1,3, JL Kupperman2, JE Stone2, PJ Kimberly1, NS Calman4, MP Hacker1, RB Birge5, TR Tritton1 and MK Newell2,3

  1. 1 Division of Immunobiology, Department of Pharmacology, Burlington, Vermont
  2. 2 Division of Immunobiology, Department of Medicine, Burlington, Vermont
  3. 3 Vermont Cancer Center, University of Vermont College of Medicine, Burlington, Vermont
  4. 4 The Institute for Urban Family Health
  5. 5 Department of Molecular Oncology, Rockefeller University, New York, New York, USA

Correspondence: Dr M Karen Newell, Division of Immunobiology, Department of Medicine, Burlington, Vermont 05405, USA.

Received 12 January 1998; Accepted 7 April 1998.



It is demonstrated that methotrexate/cisplatin-sensitive L1210 cells express low levels of major histocompatibility complex (MHC) class II relative to the high levels expressed on methotrexate (MTX)/cisplatin-resistant L1210/DDP cells. L1210 cells express cell-surface Fas, while the L1210/DDP cells express no cell-surface Fas. Expression of costimulatory molecules B7-1/B7-2 and Fas is increased on L1210 cells, but not L1210/DDP, in the presence of methotrexate or trimetrexate (TMTX). Therefore, a component of the mechanism of action of some anti-cancer agents may be to facilitate immune recognition and T cell-directed, Fas-induced cell death. Loss of cell-surface Fas expression and failure of Fas (CD95)-dependent apoptotic death has been observed when cells develop drug resistance. The defect in apoptosis can be overcome by anti-cancer agents or experimental manipulation that induce Fas expression on the drug-resistant cells.


cancer, drug resistance, Fas (CD95), immune recognition, L1210, methotrexate, MHC



Major histocompatibility complex (MHC)-encoded molecules were defined by Peter Gorer and George Snell as surface molecules responsible for rejection of tumor cells.1 These molecules are responsible for graft rejection between distinct members of the same species (`allograft rejection', reviewed in2). A part of the mechanism for both phenomena is that T cell recognition and effector function occur only when MHC molecules and antigen are engaged by the TCR.3 More recently, however, MHC molecules have been shown to function as signal-transducing receptors.4 Engagement of MHC class II results in increased expression of the molecules B7-1 and B7-2, required as costimulatory molecules for T cell activation,5 and Fas, which when engaged can result in cell death (MK Newell et al. unpubl. data).6, 7, 8 In fact, MHC class II engagement has been demonstrated to induce cell death by two distinct mechanisms, one apoptotic6, 9 and the other immune-directed osmotic rupture.7, 8 It is important to note that while MHC class II molecules are constitutively expressed on some cells, including antigen-presenting cells (APC), these molecules are inducibly expressed on most tissues under conditions of inflammation or pathology. The importance of this inducible expression is that any tissue can be recognized by immune cells and targeted for immune destruction.

Because chemotherapeutic agents induce increased Fas expression on drug-sensitive cells,10, 11 we explored the possibility that increased Fas expression may be accompanied by increased levels of MHC class II, resulting in chemotherapy enhanced immune recognition as a mechanism of action of some chemotherapeutic agents. To address these possibilities we used a leukaemic cell line known as L1210. The L1210 cells, derived from a DBA/2 mouse in 1949, are sensitive to methotrexate (MTX) and cisplatin. L1210/DDP, a drug-resistant subline which was selected in cis-diamminedichloroplatinum II (cisplatin DDP), is cross-resistant to MTX. The L1210 cell line and its sublines have been used for many years to study the mechanism of action of anti-cancer agents as well as drug resistance. In fact, our early work demonstrated the relevance of immune recognition in the mechanism of action of chemotherapeutics.12 The L1210 cells are non-adherent, morphologically round and rapidly dividing, while the resistant L1210/DDP are adherent, morphologically large and amorphous and slowly dividing cells.

Folate antagonists, such as MTX, are commonly used chemotherapeutic agents in the treatment of leukaemia, lymphoma, ovarian tumours, and squamous cell carcinoma. Drug resistance to MTX is one of the factors which limits its effectiveness as therapy for these diseases. MTX, an anti-folate oncolytic agent, competitively inhibits the binding of dihydrofolate to the enzyme dihydrofolate reductase (DHFR). Cisplatin is a metal-based oncolytic agent which binds to the nucleophilic sites on DNA, and results in changes in DNA synthesis and cell death.13 Despite the apparent dissimilarity in action of these two anti-cancer agents, collateral resistance is commonly found. The cross-resistance to MTX in L1210/DDP cells is not due to DHFR gene amplification, increased DHFR enzyme activity or decreased MTX binding to the target enzyme.14 However, the two cell lines differ in the level of tyrosine phosphorylation of a protein likely to be involved in MTX transport.15

Anti-cancer agents may work to promote the death of leukaemic cells in multiple ways. First, oncolytic agents may work by direct cytolysis. Second, oncolytic agents may promote the ability of the tumour cell to be recognized immunologically and to be killed by immune-directed cell death. Finally, oncolytic agents may work to `rewire' the death-inducing receptor–ligand pairs which include Fas and FasL. The work reported here was designed to investigate the hypothesis that anti-cancer agents alter expression of immune recognition molecules. Expression and recognition of these molecules account for part of the mechanism of action of anti-cancer agents.


Materials and methods

Cell culture and treatment

L1210 and L1210/DDP cells were cultured in RPMI supplemented with antibiotics, glutamine, 10 mmol/L HEPES buffer, 10% FCS, and 5 times 10-5 mol/L 2-mercaptoethanol. L1210 and L1210 DPP were cultured at 106 cells/well with anti-cancer agents, at the concentrations indicated, in 24-well plates overnight, unless otherwise indicated. Where indicated, the cells were cultured in folate-free RPMI (GIBCO BRL, Germantown, MD, USA) for 96 h, and treated with anti-cancer agents. Cells were then harvested by gentle pipetting and washed.

Monoclonal antibodies

The following monoclonal antibodies were purchased from PharMingen, Inc. (San Diego, CA, USA): anti-B7-1, clone 1G10; anti-B7-2, clone GL1; and anti-Fas, clone Jo2; and anti-FasL. The antibodies were either biotinylated or fluorescein conjugated for use in flow cytometric detection.

Flow cytometric analysis

Cells were resuspended in 500 muL of 5%-FCS-PBS and analysed flow cytometrically on a Coulter Profile. Cells were either stained with conjugated antibodies as indicated or were analysed immediately following the addition of 5 muL of 1 mg/mL ethidium bromide for uptake of red fluorescence. Forward angle and 90 degree light scatter were used to distinguish between live and dead cells. Dead cells were gated as forward angle light scatter low/high ethidium bromide retaining cells. Death was calculated as a percentage of the total number of cells acquired.

DNA electrophoresis

DNA was isolated and electrophoresed on 0.75% agarose as previously described.16 Fragments were visualized by staining with ethidium bromide and photographed (Polaroid, Type 57 High Speed Film).



Differential expression of MHC class II, Fas, B7-1, and B7-2 between L1210 and L1210/DDP

In order to determine the role of immune recognition molecules in the mechanisms of action of anti-cancer agents, we used flow cytometry to measure levels of expression of MHC class II, B7-1, B7-2, and Fas molecules on L1210 and L1210/DDP cells (Figure 1). As illustrated in Figure 1, L210 cells express low levels of MHC class II, moderate levels of B7-1, no B7-2, and low, but detectable, levels of Fas. In contrast, L1210/DDP cells express high levels of MHC class II, moderate levels of B7-1, very high levels of B7-2, and are negative in expression of Fas. The relative levels of expression of these molecules are established as being important in tumour rejection.17

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Flow cytometric analysis of immune recognition molecules. Levels of cell surface expression of MHC class II, B7-1, B7-2, (upper panels) and Fas (lower panels) in L1210 (a) columns and L1210/DDP (b) columns. Cells were incubated with isotype control antibodies, or fluorescein (FITC)-conjugated antibodies to MHC class II (MKD6, ref), B7-1, B7-2 (Pharmingen); or Fas (Pharmingen) as indicated. The cells were then analysed on a Coulter Elite flow cytometer.

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Chemotherapeutics induce changes in expression of MHC class II, B7-1, and B7-2 on L1210 and L1210/DDP

Given the differences in levels of expression of MHC class II, Fas, B7-1, and B7-2 between L1210 and L1210/DDP, we reasoned that one mechanism of action of anti-cancer agents may be to alter the expression of molecules recognized by immune cells, thus facilitating immune-directed cell death. We assessed changes in expression of MHC class II, B7-1, and B7-2 flow cytometrically after overnight culture of L1210 or L1210/DDP cells in the presence of anti-cancer agents, including MTX, trimetrexate (TMTX), and adriamycin. Because MTX and trimetrexate are in the family of folate antagonists, we also tested the effect of culturing the cells in folate-free medium. The absence of folic acid resulted in slowed growth and altered morphology (cells became large and adherent) of L1210 cells. The morphology and growth rate of L1210/DDP cells appeared to be unchanged by the absence of folic acid in the medium. Similarly, the absence of folic acid resulted in a relatively large increase in the level of MHC class II expression and loss of Fas expression on the L1210 cells, but not the L1210/DDP cells. Figure 2 summarizes levels of MHC class II expression, B7-1, and B7-2 expression before and after treatment with MTX, TMTX, or in the absence of folic acid. Changes in expression of B7-1 and B7-2 were drug dependent and cell-type specific. The effect of MTX on expression of molecules involved in immune recognition was inhibited in the presence of folinic acid (data not shown). These results support the hypothesis that anti-cancer agents or environmental changes alter the expression of molecules on the tumour cell, which may facilitate immune recognition.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Effect of methotrexate (MTX), trimetrexate (TMTX), and folate depletion on L1210 and L1210/DDP cells as indicated. Cells were treated in culture with (shaded square) 0.01 mumol/L methotrexate, (square with thick vertical lines) 0.1 mmol/L TMTX, and (block19;) in medium containing no folic acid for 24 h. ((filled square) no treatment.) Cells were harvested and stained with fluorescein conjugated mAbs as indicated in Figure 1. The table below the bar graphs indicates that L1210, but not L1210/DDP, loses cell-surface expression of Fas when cultured in the absence of folic acid. The data are summarized as changes in mean fluorescence intensity (MFI) over background. The data are representative of three experiments.

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Resistance to anti-cancer agent-induced apoptosis is characterized by failure to express Fas

Because it has been previously reported that some anti-cancer agents alter the expression of Fas and FasL,10, 11 we examined the possibility that the expression of Fas/FasL receptor–ligand pairs may affect, or be affected by, resistance or sensitivity in these cells. We assayed changes in expression of Fas flow cytometrically after overnight culture of L1210 or L1210/DDP in the presence of MTX or TMTX (Figure 3). Both MTX and TMTX result in induction of increased levels of Fas expression on the wild-type cells, but, in spite of the fact that at least some of the L1210/DDP cells die in the presence of the same dose of these drugs, there was no change in expression of Fas on the drug-resistant cell in the presence of anti-cancer agents. The implications of these observations and the consequences of restoration of Fas expression on resistant cell lines is discussed below.

Figure 3.
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Effect of methotrexate (MTX) and trimetrexate (TMTX) on cell-surface Fas expression of (a) L1210 and (b) L1210/DDP cells as indicated. Cells were treated in culture with (––) 0.01 mumol/L MTX or (– dotdot –) 0.1 mmol/L TMTX for 24 h. ((—) isotype; (thick line) no treatment). Cells were harvested and stained with fluorescein conjugated mAb to Fas (Jo2.2) as indicated in Figure 1.

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Cross-resistance to Fas (CD95)/FasL (CD95L) and anti-cancer agent-induced tumour cell death has been reported.11, 18 Apoptosis induced in tumour cells by cytarabine, doxorubicin, and MTX required the activation of proteases (caspases), and CD95-resistant cell lines that failed to activate caspases upon CD95 triggering were cross-resistant to drug-mediated apoptosis pathways, which led to activation of the caspases. In fact, the protease substrate poly(ADP-ribose) polymerase (PARP) is cleaved in drug-sensitive, but not drug-resistant tumour cells following drug treatment. These findings suggest that an intact Fas/FasL system may be involved in drug resistance and sensitivity, in cells derived from many tissue origins.18, 19 To determine if the lack of Fas expression on drug-resistant L1210/DDP cells is an isolated phenomenon, or representative of many resistant cell lines, we measured Fas expression on a variety of cell lines (Figure 4). The cell lines examined include L1210, a leukaemic cell; HL60, a human pro-myelocytic cell; and PC12, a pheochromocytoma cell line which can be induced to differentiate into a neuronal cell in the presence of NGF.20 Each cell line has been examined in parallel with an apoptotic resistant subline: L1210 DDP, HL60 MDR, and PC12Trk.21 L1210 DDP are resistant to cisplatin and methotrexate; HL60 MDR are resistant to adriamycin-induced apoptosis; unlike the wild-type PC12 cells, PC12 TrkA, which have been transfected with full length TrkA which results in constitutive expression of the NGF receptors, are not susceptible to apoptosis induced by NGF withdrawal.21 The apoptosis-sensitive cells from each tissue origin were morphologically round, non-adherent, rapidly dividing cells, with the exception of the PC12 cell line. The apoptosis-resistant cells from all tissue origins were morphologically large, adherent, and slowly dividing cells. The results show that cells that are apoptotic resistant express no cell-surface Fas. We found that all drug-sensitive cells examined express low, but detectable levels of Fas, while all the resistant counterparts were negative for cell-surface Fas expression (Figure 4).

Figure 4.
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Flow cytometric analysis of Fas expression. Isotype control (thick line) versus FITC-anti-Fas (Pharmingen) (thin lines), on (from top to bottom) L1210; PC12, and HL60 cells, left panels as indicated. Panels on the right are staining of resistant cell lines L1210/DDP, PC12Trk, and HL60MDR. The histograms representing isotype control (thick) versus FITC-anti-Fas (thin lines) are completely overlapping on the right panels, indicating an absence of Fas expression. A Coulter Epics Elite flow cytometer with a single excitation wavelength (488 nm) and band filters for PE (575 nm), FITC (525 nm) and Red613 (613 nm) was used to analyse the stained cells. Each sample population was classified for cell size (forward scatter) and complexity (side scatter), gated on a population of interest and evaluated using 40 000 cells. Criteria for positive staining were established by comparison with the intensity of the isotype controls (thick lines).

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Kinetics of death induction

The term `drug resistance' is commonly used to indicate that cells survive in the presence of an anti-cancer agent. One could argue that survival is a reflection of failure of the drug to kill any of the cells at a given dose of drug or survival is a result of expansion of a subpopulation of cells that survive in the presence of the agent. To distinguish between these possibilities, we treated L1210 and L1210/DDP with MTX or TMTX or we cultured the cells in medium containing no folic acid. After cells had been cultured under each condition for 24, 48, or 96 h, we monitored the cells for viability flow cytometrically (Figure 5). As indicated, the anti-cancer agents killed some cells of each type. The plateau of cell viability of the drug-resistant L1210/DDP predicts that there will be cells of this type that survive, while the growth curve of the L1210 cells clearly predicts that eventually all cells will die. The absence of folic acid prevented drug-induced death of either L1210 cells or L1210/DDP cells. Dose responses to the drugs have previously been reported.14

Figure 5.
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Effect of methotrexate (MTX), trimetrexate (TMTX), or removal of folic acid (FF) on cell viability over time. (a) L1210; (b) L1210/DDP. Cells were harvested at each indicated time point and percentage viability was calculated using flow cytometric calculations on the basis of forward scatter versus side scatter as previously described. (--square--) L1210 RPMI; (trapezoid with line) L1210 MTX; (--circle--) L1210 TMTX; (--triangle--) L1210 FF.

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Dose-dependent induction of cell death in L1210 and L1210/DDP

Based on the absence of Fas expression on the L1210/DDP cells, the reported role for Fas in apoptotic death in response to chemotherapeutic agents,10, 11 and on obvious morphological differences between the two cell types, we examined the mechanism of drug-induced cell death. As indicated in Figure 6, lanes 2, 3, 4 and 5, treatment of L1210 cells at the indicated doses of MTX results in increases in the generation of nucleosome-sized ladders of DNA, a hallmark of apoptotic cell death. At equivalent doses, lanes 4 and 7, there are nucleosomes detectable from L1210 cells, but not from L1210/DDP, respectively. It is important to note that the doses of drugs which are cytotoxic for either of the tumour cell lines in vitro are not achieved therapeutically in vivo. Figure 6 shows that the therapeutic dose of MTX, 10-8 mol/L, does not induce nucleosome-sized fragments. During chemotherapeutic treatment of patients with MTX when a concentration of 10-7 mol/L is achieved, patients are treated with leucovorin (folinic acid) to neutralize the expected toxicity of MTX (M Greenblatt pers. comm. 1998). This dose of MTX, 10-7 mol/L, is where we first see the formation of nucleosome-sized fragments in vitro in the drug-sensitive, but not drug-resistant, cells. This, along with our data which show alterations in immune recognition molecules during drug treatment, supports the notion that anti-cancer agents induce cytotoxicity of tumour cells in vivo by a concert of drug action and immune recognition and that the levels of drug concentration that can be used therapeutically are insufficient for cytotoxicity.

Figure 6.
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L1210, lanes 1– 5, and L1210/DDP, lanes 6–10, were cultured in the presence of methotrexate (MTX) (L1210 cells: lane 1, no treatment; lane 2, 10-9 mol/L; lane 3, 10-8 mol/L; lane 4, 10-7 mol/L; lane 5, 10-6 mol/L; L1210/DDP cells: lane 6, no treatment; lane 7, 10-7 mol/L; lane 8, 10-6 mol/L; lane 9, 10-5 mol/L; lane 10, 10-4 mol/L) for 48 h. Cells were harvested, lysed in NP-40 as described and fragmented DNA analysed by agarose gel electrophoresis.

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Treatment of L1210/DDP cells with staurosporin restores Fas expression and apoptosis

L1210, but not L1210/DDP, cells appear to undergo apoptotic cell death at the described concentrations of MTX. We addressed the possibility that the failure of L1210/DDP to express Fas might be related to failure to undergo apoptosis. We treated L1210 or L1210/DDP cells with staurosporin, which has been reported to induce the death of some tumour cells by apoptosis. Staurosporin induced apoptosis of L1210 cells, but not L1210/DDP. Staurosporin induced increased Fas expression on L1210 and L1210/DDP (Figure 7). In the presence of staurosporin, L1210/DDP changed morphologically and began to divide rapidly. The change in phenotype of L1210/DDP is consistent with a reversion back to the phenotype of the L1210 cells and, in fact, while culture with staurosporin did not kill the cells, they became sensitive to MTX-induced apoptosis. These results suggest that while the L1210/DDP cells are Fas-, MTX and TMTX resistant, and apoptotic defective, the cells can revert to Fas+, MTX sensitive, and apoptotic in the presence of staurosporin. We are investigating the mechanism for the drug- or staurosporin-induced reversion.

Figure 7.
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(a) L1210 and (b) L1210/DDP cells were harvested after 24 h culture in the presence or absence of staurosporin at 5 ng/mL. Cells were stained as described and analysed flow cytometrically as mean fluorescence intensity (MFI) over background as indicated. The data are representative of three experiments. (shaded square) ADR, adriamycin; (square with 315 degree lines) staurosporin.

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Treatment of MTX-sensitive L1210 cells, which express low levels of MHC class II, and MTX-resistant L1210/DDP cells, which express relatively high levels of MHC class II molecules, with MTX, TMTX and adriamycin, results in increased or decreased expression of MHC class II, respectively. While the L1210 cells are constitutively Fas+ and the L1210/DDP cells are constitutively Fas-, Fas expression increases on L1210 and is induced on L1210/DDP cells, in the presence of agents to which each cell line is sensitive. The defect in apoptosis of the L1210/DDP cells can be reversed by incubation with staurosporin and the change is accompanied by re-expression of Fas on cells which were previously Fas-. These results suggest that Fas expression may be a reflection of drug sensitivity. We suggest that the mechanism of action of MTX and TMTX is to induce expression of immune recognition molecules, such as MHC class II, to induce expression of Fas, and together to facilitate drug-induced, immune-mediated tumour cell destruction.

The centrally important function for MHC class II molecules as molecules which restrict the activation of antigen-specific T cells remains unchallenged. MHC molecules also act as signal transducing receptors and engagement of MHC class II can result in apoptotic cell death.6 The mechanism of MHC class II-mediated death involves Fas and its ligand.8 MHC class II engagement also results in increased expression of Fas, B7-1, and B7-2.5, 22 Taken together, these reports provide evidence that MHC engagement can result in immune-directed cell death.

There have been paradigms in both immunology and oncology which the present report brings into question. First, a paradigm of modern immunology has been that MHC-encoded class II molecules bind and present antigenic peptides to helper T cells during an immune response. While this has proven to be true, there has been growing acceptance that MHC class II molecules are capable of transducing signals to the cells on which they are expressed and that the signals depend on the state of activation and, possibly the cell-cycle state of the cell. Ligation of MHC class II results in increases in cyclic AMP (cAMP),4 alterations in levels of intracellular Ca2+,23 activation of tyrosine kinases,24 and changes in intracellular ornithine decarboxylase.25 Importantly, MHC class II engagement has been demonstrated to induce cell death by either of two mechanisms, one apoptotic and the other immune-directed osmotic rupture.6, 7, 8 The signals delivered by MHC engagement and whether the signals will result in cell death depend on the cell-cycle state, the tissue expressing the MHC class II molecule, and the presence of growth factors or growth signals.5, 6, 17 In this report, we observed that the levels of MHC class II expression change in response to the changes in levels of folic acid and in response to treatment with anti-cancer agents. If these changes act to alter the cell-surface expression of molecules necessary to relay information to cells in the immune system, the changes in expression and `rewiring' may have evolved as an indicator of damage and to direct the repair or demise of the cells at risk. The results of the present report would suggest that in fact this is the case for tumour cells and support the notion that immune recognition may be important in the mechanism of action of anti-cancer agents.

The second paradigm is that oncolytic agents induce death of tumour cells by direct cytolysis. This and other reports have established that anti-cancer agents also alter the levels of expression and the function of molecules recognized by the immune system and known to be involved in cell death, the Fas/FasL receptor–ligand pairs.10, 11 Taken together with the notion that oncolytic agents may promote the ability of the tumour cells to be recognized immunologically, these results suggest that tumour cells are killed by immune-directed cell death. Furthermore, oncolytic agents may work to `rewire' the death-inducing receptor–ligand pairs which include Fas and FasL such that they support cell-cycle dependent growth and, when interrupted, apoptotic, cell-cycle dependent death. We hypothesize that Fas/FasL-mediated apoptosis is really abortive Fas/FasL- associated cell division and that a tumour cell is sensitive to agents which induce apoptosis when those agents or therapeutic manipulations induce the expression of Fas on a Fas- cell or FasL on a Fas+ cell which results in abortive cell division in the presence of the drugs. This hypothesis is supported by data showing that culture of L1210 cells in folate-free medium induces growth arrest, increased expression of MHC class II molecules, loss of Fas expression, and resistance to MTX, TMTX and adriamycin. Experiments are currently underway in our laboratory to address the possibility that other anti-cancer agents exert similar changes in immune recognition molecules and the consequent immune-directed growth is aborted in the presence of chemotherapeutic regimens such that the final outcome is tumour cell death.



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This work was supported by grants ACS DHP-170 (AB), ACS (Vermont division) (AB), RO1 AI 33470 (MKN) and University of Vermont College of Medicine (MKN).