Alterations in target cell membrane phospholipids alter T cell but not NK cell killing

Immunobiology 218 (2013) 21–27

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Alterations in target cell membrane phospholipids alter T cell but not NK cell killing David T. Harris ∗ Department of Immunobiology, College of Medicine, The University of Arizona, AHSC 6122, PO Box 245221, Tucson, AZ 85724, United States

a r t i c l e

i n f o

Article history: Received 29 September 2011 Received in revised form 18 January 2012 Accepted 21 January 2012 Keywords: CTL Cytolysis NK Phospholipids Recognition

a b s t r a c t The ability of tumor cells to grow progressively in vivo despite the host immune response remains a major conundrum in tumor immunology. Various mechanisms have been proposed to explain how tumors evade immune destruction. The work presented herein shows that simple alterations in plasma membrane phospholipid composition can alter susceptibility to immune lysis. The phospholipid composition of target cells was specifically altered by growth in medium containing choline analogs. Manipulation of membrane phospholipids was observed to alter cell susceptibility to murine CTL but not NK cell lysis. The effects of such changes in phospholipid composition on CTL-mediated lysis appeared to occur during the recognition phase of lysis. This mechanism could be a means by which tumor cells, as well as other pathogenic organisms, escape immune detection and destruction. © 2012 Elsevier GmbH. All rights reserved.

Introduction The ability of tumor cells to grow progressively in vivo despite the host’s immune responses against it remains a significant issue in tumor immunology. Immune surveillance mechanisms which must be circumvented include tumor-specific cytotoxic T lymphocytes (CTL; Alexander et al. 1966), tumor-specific antibodies, natural killer (NK) cells, and antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by macrophages and NK cells (Alexander et al. 1966; Allavena et al. 1981; Baldarin and Barker 1967; Berke and Amos 1973; Blank et al. 1976; Engers and Unanue 1974; Fulton and Levy 1980; Hellstrom and Hellstrom 1971, 1977; Herberman 1980; MacDonald et al. 1974; Mathe et al. 1969; Nowell 1976; Roelants and Askonas 1971; Schecter et al. 1976; Ting and Law 1967). In addition, many tumors display altered metabolic profiles which may account for some of the ability to escape immune destruction (Mandel and Clark 1978; Schlager and Ohanian 1980; Schlager 1982; Shinitzky et al. 1979). Altered metabolism may include changes in cell cycle status (Leneva and Svet-Moldavsky 1974), down-regulation of tumor and histocompability antigens (Berke et al. 1981) or modification of cellular repair mechanisms

Abbreviations: AB, cells grown in l-2-amino-butanol; AP, 3-aminopropanol; C, choline; Con A, concanavalin A; CTL, cytotoxic T lymphocyte; DME, dimethylethanolamine; E, ethanolamine; MHC, major histocompatibility complex; MME, monomethylethanolamine; NK, natural killer; PEL, peritoneal exudate cells; TLC, thin layer chromatography. ∗ Corresponding author. Tel.: +1 520 626 5127; fax: +520 626 2100. E-mail address: [email protected] 0171-2985/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2012.01.017

(Ackerstaff et al. 2003; Podo et al. 2011; Schlager 1982). A simple mechanism previously hypothesized to be able to affect many of these parameters is alteration of the membrane lipid composition (Ferguson et al. 1975; Glaser et al. 1974; Mandel and Clark 1978; Schlager 1982; Schlager and Ohanian 1980; Shinitzky et al. 1979). Mandel and Clark (1978) altered the fatty acid composition of tumor cells in order to change the fluidity of the plasma membrane but failed to observe any changes in either antibody plus complement (Ab) or CTL-mediated lysis of the cells. Shinitzky et al. (1979) changed the membrane viscosity of tumor cells by addition (or depletion) of cholesterol or stearic acid and found that when the viscosity of the plasma membrane increased the cells became better immunogens. Also, Schlager and Ohanian (1980) and Schlager (1982), using a system of metabolic inhibitors, altered the susceptibility of tumor cells to Ab- and CTL-mediated lysis; correlating their results with the ability of the target cells to incorporate fatty acids into complex membrane-bound lipids. Each of these studies has provided useful information regarding tumor cell variables which may affect susceptibility to lysis but each had two significant limitations. First, the studies employed manipulations which may change more than just the parameter under investigation. Second, manipulation of specific parameters (such as just one particular fatty acid or only one phospholipid) could not be achieved. The objective of the work presented herein was to determine if simple and specific alterations in target cell plasma membrane phospholipid composition could alter cellular immune responses. Cells were grown in medium containing specific choline analogs to induce specific plasma membrane phospholipid alterations (Ferguson et al. 1975; Gilmore et al. 1979; Glaser et al. 1974). It was observed that significant changes in target cell susceptibility

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to lysis by CTL, but not by NK, were induced. It is proposed that membrane phospholipid composition may significantly alter a target cell’s susceptibility to immune destruction. Materials and methods Mice The following strains of inbred mice were used in these studies: Balb.b, Balb.k, C57Bl6 and C3H/HeJ. These mice were obtained from breeding stocks originally obtained from Dr. H. Eisen (MIT) or Dr. D.B. Amos (Duke University). All animal studies were reviewed, approved and conducted according to AAALAC guidelines.

methanol and 0.2 ml of H2 SO4 to samples in Teflon-lined screwcapped tubes. The tubes were then flushed with nitrogen, sealed and heated at 80 ◦ C for 2.5 h after which they were cooled, 2.0 ml of H2 O and 2.5 ml of pentane was added, and the pentane layer removed for analysis of fatty acid content via gas layer chromatography (OV351 capillary column, 62 m in length, 1 ml/min Helium flow rate, 155–200 ◦ C at 8 ◦ C/min and 201–245 ◦ C at 5 ◦ C/min). Production of concanavalin A supernates Concanavalin A (Con A) supernates were generated by culture of Balb.b splenocytes (3 × 106 cells/ml) in RPMI medium containing 2 ␮g/ml Con A. After 24 h the cells were removed by centrifugation and supernates sterilized by filtration (0.2 ␮).

Cell culture Production of cytotoxic T lymphocytes (CTL) The following cell lines were used in these studies: LM (a fibroblast of C3H origin), YAC-1 (a lymphoma of ASN origin), and RDM-4 (a lymphoma of AKR origin). YAC-1 and RDM-4 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), glutamine and antibiotics. Phospholipid supplementation of cells Cultures of LM cells were grown at 37 ◦ C in 75 cm2 tissue culture flasks in 10 ml of Higuchi’s medium (Higuchi 1970) containing 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer, pH 7.4. This medium is a chemically defined, lipidfree medium that supports growth of LM cells indefinitely in the absence of serum when choline (C) is present in sufficient concentration (Higuchi 1970). Cells to be grown in the presence of C analogs were harvested by pipetting, washed twice with PBS and resuspended in the desired medium. The following C analogs were utilized: ethanolamine (E), monomethylethanolamine (MME), dimethylethanolamine (DME), l-2-amino-1-butanol (AB) and 3aminopropanol (AP). All analogs were added at a concentration of 40 ␮g/ml and except for AB, were obtained from Eastman Kodak Company. AB was obtained from Aldrich Chemical Company. The cells were grown for 3 days in the supplemented medium prior to use in the various assays (Glaser et al. 1974). Phospholipid and fatty acid analyses Cells grown in choline were incubated for 5 days in the presence of 5 ␮Ci/ml of 32 P-phosphate (as orthophosphate) prior to exposure to C analogs. Cells were then exposed to the various C analogs for 3 days as previously described, keeping the 32 P concentration constant throughout the time period. At the end of this time the cells were washed twice by centrifugation in Ca2+ , Mg2+ -free PBS and whole cell lipids extracted by the method of Bligh and Dyer (1959) as described by Ames (1968). The phospholipids were separated by 2-dimensional thin layer chromatography (TLC) on silica gel H (250 ␮m), made visible by autoradiography (Ames 1968), scraped and counted in Omnifluor-toluene–Triton X-100–H2 O (4%-2:1:0.2) scintillation fluid in a Packard scintillation counter. Phospholipids were identified by comparison with known standards. The solvent system consisted of chloroform–methanol–H2 O (87:31:5) in the first dimension and N-butanol–glacial acetic acid–H2 O (80:26:26) in the second dimension. For fatty acids analysis, cells were harvested and phospholipids extracted as above. The phospholipids were then separated from the neutral lipids on a silicic acid column (0.3 g Unisil in a disposable Pasteur pipette plugged with glass wool). The neutral lipids and phospholipids were eluted with 5 ml of chloroform and 4 ml of methanol, respectively. Fatty acid methyl esters were prepared from the phospholipid fraction by the addition of 2.5 ml of pure

Anti-H-2k CTL were prepared by one-way mixed lymphocyte reactions (MLR). Briefly, 1 × 108 viable splenocytes from Balb.b or C57Bl/6 mice (responders) were incubated with 5 × 107 irradiated (1000 rads) or mitomycin C-treated (Sigma, 50 ␮g/ml, 1 h at 37 ◦ C) Balb.k splenocytes (stimulators) in upright 75 cm2 tissue culture flasks in 50 ml media to which 10% Con A supernates, 5 × 10−5 M 2mercaptoethanol and 1% sodium pyruvate was added. The effector cells were harvested after 5–7 days of incubation. In some experiments, peritoneal exudate lymphocytes (PEL) were used as a source of CTL. PEL were obtained after intraperitoneal injection of 2 × 107 RDM-4, YAC-1, LM or Balb.k splenocytes into Balb.b or C57Bl/6 mice. After 10–12 days the mice were euthanized and PEL harvested from the peritoneal cavity with a 10 ml syringe. Natural killer (NK) cells NK cells were elicited by intra-cardiac injection of 1 × 106 pfu of vesicular stomatitis virus (VSV) into mice of the indicated strains. 1–2 days later spleens were removed and single cell suspensions were used as a source of activated NK cells. CTL and NK assays Target cells were labeled with 51 Cr-sodium chromate and mixed with effector cells in 96 well microtitre plates for 6 h at 37 ◦ C. Cytotoxicity assays were stopped by addition of cold PBS and centrifugation. Radioactivity in the supernatants and pellets was determined by use of a Beckman gamma counter. Controls included targets alone. Data are shown corrected for the spontaneous 51 Cr release of each target cell. Target cell susceptibility to T cell lysis The protocol of Berke et al. (1986) was used to study target cell susceptibility to T cell lysis. Briefly, antigen-non-specific lytic T cells were generated by culture of splenocytes for 3 days with Con A (5 × 106 /ml, 2 ␮g/ml Con A) in RPMI media. Prior to the killing assays, target cells were pre-coated with 10 ␮g/ml Con A at 37 ◦ C for 30 min, washed extensively and resuspended in RPMI media. Effector and target cells were then admixed and incubated at 37 ◦ C for 4 h in the presence of 10 ␮g/ml Con A. Conjugate formation of effector and target cells was also performed under identical conditions (see below). Conjugate assays Determination of effector cell binding to various target cells was performed by the method of Gilmer et al. (1978). Briefly, 1 × 106

D.T. Harris / Immunobiology 218 (2013) 21–27

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Table 1 Phospholipid composition of target cells grown in choline analogs. Supplementa

C E MME DME AB AP

Phospholipid composition (%)b

Total %c

PC

PE

PMME

PDME

PAB

PAP

SM

PS/PI

CL

49.8 44.2 20.1 13.0 39.1 25.8

25.4 32.5 13.5 15.3 16.1 14.8

1.8 – 44.6 1.8 0.7 1.5

2.1 1.2 3.8 53.4 0.7 –

– – – – 22.5 –

– – – – – 34.9

10.3 9.1 9.6 7.7 10.6 10.8

7.4 9.2 5.8 6.5 7.6 8.5

3.1 3.9 2.6 2.4 2.7 3.7

99.9 100 100 100.1 100 100

a Cells were grown for 3 days in medium containing 40 ␮g/ml of either choline (C), ethanolamine (E), monomethylethanolamine (MME), dimethylethanolamine (DME), 1-2amino-butanol (AB), or 3-amino-propanol (AP). Cells were 32 P-labeled and analyzed by TLC as described in Section “Materials and methods”. Data represent the combination of three individual experiments and are expressed as percent composition. Standard deviations between experiments for an individual phospholipid class differed by less than 10%. b Cellular membranes were analyzed for the following lipid classes: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PMME), phosphatidyldimethylethanolamine (PDME), phosphatidyl-1-2-amino-butanol (PAB), phosphatidyl-3-aminopropanol (PAP), sphingomyelin (SM), phosphatidylserine/phosphatidylinositol (PS/PI), and cardiolipin (CL). c Total counts per plate for the 3 experiments ranged between 288,817 (experiment 1) and 2,581,792 (experiment 3) counts.

target cells were mixed with 1 × 106 effector cells in 1.0 ml of icecold RPMI media. The cell mixture was centrifuged at 250 × g for 1 min and allowed to incubate for 15 min at 23 ◦ C. At that time the cell pellet was gently resuspended with a Pasteur pipette, placed on a hemocytometer, and the cover slip gently tapped to disperse spontaneous conjugates. The number of conjugates was then determined, with at least 250 target cells counted per condition. Identification of the conjugates was facilitated by the large size of the target cells in relation to the effector cells. In addition, conjugates were counted for target cells alone and effector cells alone under identical conditions. FACS analysis Levels of detectable H-2 major histocompatibility complex (MHC) antigens were analyzed by FACS analysis. The anti-H-2Kk mAb (clone 15.3.1; ATCC) and a FITC-labeled rabbit-anti-mouse IgG antibody (Cappel Laboratories) were used for this purpose. Samples were analyzed using an Ortho 50H Cytofluorgraph equipped with a 4W argon laser. Statistics All statistical analyses were performed using Student’s t-tests (significance set at p < 0.05) as found in SPSS.

desired specific changes in plasma membrane phospholipid composition. Choline-supplemented media served as the control media and was used for long-term maintenance of the cells. As shown in Table 1, these culture conditions resulted in significant and specific alterations in the phospholipid profile of the target cells. Supplementation of the media with a specific C analog (i.e., phospholipid precursor) resulted in that single phospholipid becoming, if not the major, one of the major phospholipid constituents of the target cells. In the cases of AB and AP supplementation, it was possible to introduce a non-naturally occurring phospholipid component into the cellular phospholipid profile to significant proportions. No significant differences were found in terms of the minor phospholipids (e.g., sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin). Thus, the experimental protocol allowed for specific alterations in specific phospholipid components of the target cells. In addition, analyses were performed to determine if these modifications significantly affected the fatty acid composition of the cells, as this variable has been shown to affect target cell lysis (Verkamp et al. 1962). As shown in Table 2, no significant changes in the majority of the fatty acid composition due to phospholipid alterations were observed, although there was a statistically significant difference in some cells for the 16:1 fatty acids. Fatty acids of the 18:1 variety were the major constituent (>50%) in each one of the analog-supplemented cells. Only minor variations in other fatty acids (16:0 and 18:0) were observed.

Results Choline analog supplementation specifically alters target cell membrane phospholipids Target cells were grown in the presence of choline or one of five analogs (E, MME, DME, AB or AP) in order to achieve the

Effects of membrane phospholipid alterations on susceptibility to immune lysis The effects of phospholipid manipulation on susceptibility of target cells to CTL- and NK-mediated cytolysis were examined. The susceptibility of target cells to CTL-mediated lysis after growth in

Table 2 Effect of phospholipid supplementation on fatty acid composition. Cell

Fatty acid analyses

Media

16:0

16:1

18:0

18:1

18:2

18:3

22:5

22:6

C E MME DME AB AP

17.6 10.2 11.2 10.0 10.0 14.4

3.3 3.4 a 13.9 a 13.0 a 13.0 a 13.8

12.8 17.2 18.7 17.1 14.4 20.1

58.9 57.4 53.1 50.7 63.4 55.9

0.0 0.0 2.4 3.2 0.0 0.0

0.0 0.0 0.0 2.0 0.0 0.0

1.3 1.8 2.5 3.4 2.4 1.4

3.0 4.6 2.5 3.4 5.0 3.4

Cells were grown in either choline (C) or one of the analogs (E, MME, DME, AB or AP) at 40 ␮g/ml for 3 days as described in Section “Materials and methods” prior to analysis. Fatty acid composition analysis was performed as described in Section “Materials and methods”. That is, fatty acids were separated by gas layer chromatography and identified by co-retention times with known standards. Data represent the percent composition for each individual cell type. Data are representative of 3 independent experiments. a Indicates significant difference as compared to the (C) condition at p < 0.05.

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Fig. 1. Antigen-specific cytotoxic T lymphocyte (CTL)-mediated lysis of target cells with altered phospholipid compositions. Assays were performed as described in Section “Materials and methods”. Data are presented as the mean ± SD of 5 independent experiments for effector:target cell (E:T) ratios of 25:1 and 200:1. All conditions were significantly different from C at p < 0.05. C, MME and DME targets were significantly different (p < 0.05) from E, AB and AP targets, as groups. The spontaneous release of 51 Cr-radiolabel observed in each of the cytotoxicity assays was low and comparable between the various target cells (15–25%).

the C analogs is shown in Fig. 1. Data are presented for two different effector cell to target cell (E:T) ratios to better emphasize the effects of phospholipid composition on cytotoxicity. Cells grown in the C, MME and DME supplemented media were found to be very susceptible to CTL-mediated cytolysis, while the E, AB and AP supplemented target cells were quite resistant to lysis (e.g., see 25:1 ratio; p < 0.05 significance). In contrast to the CTLs, NK-mediated cytotoxicity was not significantly affected by any of the modifications in target cell phospholipid composition (Fig. 2). Each of the C analogs resulted in target cells displaying equal susceptibility to NK-mediated lysis. The levels of overall lysis were low (20–25%) but were characteristic of the levels of killing obtained when fibroblast are used as targets in NK killing assays (Lindsay and Allandyce 1982).

Fig. 2. Natural killer (NK) cell-mediated lysis of phospholipid-altered target cells. A 6-h cytotoxicity assay was performed as described in Section “Materials and methods”. Data are presented as the mean ± SD for 6 independent experiments for an E:T ratio of 200:1. The spontaneous release of 51 Cr-radiolabel observed in each of the cytotoxicity assays was low and comparable between the various target cells (15–25%).

However, with lectin-dependent CTL assays it is possible to form an equal number of effector cell/target cell conjugates regardless of the effects of media supplementation. This approach allowed for an analysis of the lytic stage of CTL killing independently of target cell recognition. As can be observed in Table 3, regardless of the target cell’s susceptibility to antigen-specific CTL-mediated lysis (DME or MME vs. C vs. E, AB or AP) CTL induction of a lytic lesion was comparable. Thus, alteration of target cell phospholipid composition did not seem to alter the inherent susceptibility of target cells to CTL-induced cell damage.

Possible mechanisms of plasma membrane phospholipid alterations in CTL lysis Efforts were undertaken to identify the mechanism by which specific plasma membrane phospholipid alterations altered CTL lysis. Similar results (although somewhat more striking in comparison) were observed when CTL-mediated lytic kinetics were analyzed (Fig. 3). Once again, cells grown in C, DME and MME were significantly more susceptible to lysis than target cells grown in either AP or AB, especially at later time points. Thus, the previously observed effects on T cell killing were not merely due to “point-in-time” observations. To assess if the altered target cells displayed an inherent resistance to T cell-mediated killing the susceptibility of each target cell to induction of the CTL “lethal hit” was analyzed. We used the method of Berke et al. (1986), employing antigen-non-specific 72 h Con A blasts or MLR-generated lymphocytes as a source of effector cells, in cytolytic assays performed in the presence of Con A. This assay is dependent upon a similar recognition stage of the CTL-mediated mechanism of lysis as with antigen-specific CTL, including a dependence on the presence of H-2 antigens.

Fig. 3. Kinetics of CTL-mediated lysis of phospholipid altered target cells. Each determination was made in triplicate and data are presented for lysis obtained with E:T ratios of either (A) 33:1 or (B) 100:1. The spontaneous release of 51 Cr-radiolabel observed in each of the cytotoxicity assays was low and comparable between the various target cells (15–25%).

D.T. Harris / Immunobiology 218 (2013) 21–27

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Table 3 Susceptibility of phospholipid-altered target cells to non-specific T cell lysis. Target cella

Effector cell sourceb

Percent conjugatesc

Balb.b

C E MME DME AB AP

C3H.HeJ

Balb.b-anti-CBA

3:1

50:1

3:1

50:1

3:1

50:1

10.0 30.0 20.7 16.9 28.3 31.8

61.5 57.0 56.6 55.7 75.0 78.6

4.5 14.1 3.2 11.2 20.0 17.7

50.0 65.8 58.9 51.4 68.4 74.6

4.5 4.4 5.3 7.1 17.6 15.9

38.0 58.4 45.1 54.3 55.8 55.7

10.2 12.6 12.3 12.2 10.9 10.5

Cells were grown for 3 days in medium containing 40 ␮g/ml of either choline (C) or analogs (E, MME, DME, AB or AP) as described. Effector cells were derived either by one-way MLR (Balb.b-anti-CBA) or from 72 h ConA blasts (Balb.b and C3H.HeJ). Data are presented as the percent specific 51 Cr release for E:T ratios of 3:1 and 50:1. c 1 × 106 CTL were mixed with 1 × 106 target cells in 1.0 ml of complete medium, centrifuged at 250 × g for 1 min, and incubated at 23 ◦ C for 15 min. Data represent percent conjugates formed per 200 target cells counted. Target cells were pre-incubated with 10 ␮g/ml ConA for 30 min at 37 ◦ C. Conjugate formation was performed in the presence of 10 ␮g/ml ConA. a

b

Table 4 Effects of phospholipid composition on target cell: CTL conjugate formation. a

Supplement

C E MME DME AB AP

Percent conjugatesb Experiment 1

Experiment 2

Experiment 3

Experiment 4

Experiment 5

10.8 15.5 16.4 17.6 6.6 8.0

20.2 19.6 17.6 32.6 14.0 13.4

11.8 15.1 16.0 17.8 6.2 4.4

21.2 26.1 23.3 31.0 16.3 16.5

10.2 12.1 15.7 23.3 7.2 7.9

In each of the experiments shown, the group composed of C, E, MME and DME was found to be significantly different from the group containing AB- and AP-supplemented cells (p < 0.05). a Cells were grown for 3 days in medium containing 40 ␮g/ml of choline (C) or analog (E, MME, DME, AB or AP) as described. b 1 × 106 target cells were mixed with 1 × 106 CTL in 1.0 ml complete medium on ice. Cells were pelleted (250 × g, 1 min) and incubated at 23 ◦ C for 15 min. Cells were then resuspended and conjugates counted. At least 250 target cells were counted. CTL were derived from either one-way MLR lymphocytes or PELs (experiments 2 and 4) as described in Section “Materials and methods”.

The levels of MHC (H-2) antigen expression were then examined. Cells grown in C, DME and AP were chosen for analysis in that these conditions represented the entire range of lytic susceptibility. As seen in Fig. 4, alterations in target cell phospholipid composition

significantly affected the levels of detectable H-2 antigens, as well as the percentage of cells in each population expressing MHC molecules. Target cell susceptibility to CTL-mediated lysis was found to be correlated to the percentage of target cells in the total cell population that expressed a high level of H-2 molecules (greater than 400 fluorescence units). Thus, DME-supplemented cells (a highly susceptible target) had greater than 50% of the cell population exhibiting very high levels of H-2 antigens, while APsupplemented cells (a highly resistant target) was observed to have only approximately 5% of its cell population possessing a high level of MHC antigens. The C-supplemented cells (normal conditions), intermediate in terms of susceptibility to CTL-mediated killing, were also intermediate in the percentage of the cell population with high levels of H-2 antigens (approximately 33%). These observations led to an examination of the effect of changes in membrane phospholipid composition on the ability of the antiH-2k specific CTL to form conjugates with the various (H-2k ) target cell populations (see Table 4). Target cells which had been found to be very susceptible to CTL-mediated lysis (e.g., C, MME and DME) were also found to be those cells which formed the greatest number of conjugates, with the exception of E-supplemented cells. This observation was in concordance with the observations previously noted for H-2 antigen levels and susceptibility to CTL-mediated lysis.

Discussion Fig. 4. Effects of phospholipid alterations on H-2 antigen expression. Expression of H-2 antigens on target cells was made by flow cytometric analysis using an antiH-2Kk mAb (clone 15.3.1) and a FITC-labeled rabbit-anti-mouse IgG antibody, as described. Cells were grown in medium (3 days) containing either (A) choline (C); (B) dimethylethanolamine (DME); or (C) 3-aminopropanol (AP). All three analyses are shown in (D) for comparison (smoothed curve comparisons).

Immune responses to tumors in animals previously immunized with non-viable tumor cells are well established (Alexander et al. 1966; Baldarin and Barker 1967), and there is little doubt that malignant cells can be destroyed in vitro by different cellular

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effector mechanisms. The mechanism(s) responsible for the ability of malignant cells to survive, exhibit progressive growth, and metastasize in vivo remains a mystery in many instances. As neoplasms frequently arise as a clone from a single cell (Nowell 1976), any new or modified genetic information conferring selective growth advantages may be preferentially selected and maintained. These cells can then give rise to a dominant phenotype of the newly emerging malignancy, potentially bypassing the host’s defenses. Various mechanisms proposed to overcome immune defenses have included the generation of “blocking” antibody (Engers and Unanue 1974; Hellstrom and Hellstrom 1971, 1977), antigen shedding by the tumor (Roelants and Askonas 1971), and tumor-induced host suppression (Allavena et al. 1981; Fulton and Levy 1980). There are three basic “types” of tumor mechanisms that may affect host immune defenses: lack of recognition of the tumor by the immune system; lack of interaction of the tumor with the host’s various cytolytic machinery; and innate resistance of the tumor to immune cytolysis. Cellular immune mechanisms that must be circumvented include tumor-specific CTL and NK cells; both of which have received considerable attention with regard to their role in tumor immunity (Berke and Amos 1973; Blank et al. 1976; Herberman 1980; MacDonald et al. 1974; Mathe et al. 1969; Schecter et al. 1976; Ting and Law 1967). Previous investigators have attempted to correlate the susceptibility of tumor cells to immune lysis with alterations in a variety of physical parameters of the tumor cells. Unfortunately, in these earlier studies it was not possible to alter a single specific cellular component independently of other cellular variables. Herein, we have utilized a model in which the effect of specific alterations in the phospholipid composition of a target cell could be analyzed for its contribution to the susceptibility of the target cell to immune lysis (Ferguson et al. 1975; Gilmore et al. 1979; Glaser et al. 1974). Changes in phospholipid composition were found to result in significant differences in susceptibility of the target cells to CTL-, but not NK-mediated killing. To ascertain the mechanism behind our observations the recognition and lytic stages of the CTL killing mechanism were examined. The recognition stage refers to the ability of the CTL to identify and bind to the target cell. The lytic phase refers to the steps involved in the induction of a functional lesion in the cytoplasmic membrane of the target cell, characterized by the subsequent disintegration of the target cell. It was observed that phospholipid compositional changes affected the recognition stage of the CTL killing mechanism, but not the lytic phase. The changes in the recognition events of this initiating phase of CTL-mediated lysis were reflected in the ability of the effector populations to form conjugates with the various target cells. The ability to form conjugates was found to be correlated with cellular levels of H-2 antigens. H-2 antigens, while involved in target cell recognition, may also act to increase the percentage of functional conjugates formed (Berke et al. 1981, 1986). It was interesting to observe that changes in phospholipid composition drastically affected the expression and distribution of H-2 antigens on target cells. To our knowledge this finding is the first demonstration of such a phenomenon. Overall, it seemed that once the CTL and target cell managed to interact (i.e., recognition occurred) the following steps involved in lysis were equivalent. Thus, the data suggested that the limiting step in the CTL lytic mechanism was related to the binding avidity of the CTL to the target cell. Specific phospholipid alterations caused target cells to display higher levels of H-2 antigens and to become more susceptible to CTL-mediated lysis (DME > C > AP). This observation was correlated with increased conjugate formation. Thus, as might be expected, higher levels of H-2 antigens may have promoted and increased recognition of the target cells. However, the ethanolamine (E)-supplemented target cells were an exception to this finding. E-target cells were somewhat resistant

to lysis but formed high numbers of conjugates with CTL. The exact reason behind these observations is not known. However, it may be due to the relatively poor incorporation of E into the plasma membranes resulting in a mixed “phenotype”, or some other mechanism may be involved. Further experimentation is needed to resolve this discrepancy. It was of considerable interest that the changes in phospholipid composition which affected the CTL did not alter NK-mediated cytotoxicity. The failure to detect any alteration in NK lysis was not due to an insensitive assay system (i.e., using fibroblasts as target cells), as levels of specific lysis of 25% should be sufficient to observe either a twofold increase or decrease in the susceptibility of a cell to killing. This lack of alteration in NK cell lysis may be a significant observation in terms of the NK cell’s role in the detection and destruction of developing tumors. If indeed NK cells act as the first line of defense against malignancy as has been suggested (see e.g., Herberman 1980), then the host would benefit by possessing a cytolytic mechanism that was not affected by changes in this particular characteristic of a tumor cell, and still be able to effect its destruction. In conclusion, the purpose of the study presented herein has been to analyze the role of membrane phospholipid composition in a cell’s susceptibility to immune cytolysis. This aspect was emphasized because it is at the level of the cytoplasmic membrane that target structures recognized by these cytolytic systems are expressed, this is the location where the target cell/effector cell interaction is effected, and it is the location at which the cytolytic event is imparted. Thus, any alteration in determinants expressed at the level of the plasma membrane should have a considerable effect on the processes of immune destruction. Simple and specific modifications in phospholipid composition of a target cell could affect the lytic processes of these effector systems. Berke et al. (1978) had previously speculated that membrane phospholipids might be of some importance in terms of CTL lysis, in that it might promote the CTL/target cell interactions necessary for killing. This speculation proved to be correct in that we observed that simple changes in plasma membrane phospholipid composition impaired the ability of a target cell to be recognized by effector T cells. The membrane phospholipid composition may not only affect anti-tumor immune responses (Ackerstaff et al. 2003; Iorio et al. 2005; Robinson et al. 2001) it theoretically may also affect the ability of the host to generate cytolytic responses to viral, bacterial and/or parasitic infections due to a lack of recognition (Alving et al. 2006; Bansal et al. 2005; Sinibaldi et al. 1992). The most difficult question to answer in this setting is whether such changes in membrane phospholipids occur naturally in vivo. Although there is in vivo evidence as discussed below, this aspect of immunological deviation has never been reported previously. Most investigators have focused on the genetic mechanisms used to decrease MHC expression by viruses and tumors, rather than considering whether simple metabolic changes could induce the same effects (and may function in concert with these genetic mechanisms). However, Ackerstaff et al. (2003) upon reviewing the role of phospholipid metabolism in cancer cells and tumors, made a case for targeting the aberrant phospholipid metabolism of cancer cells. The elevation of phosphocholine (PC) and total choline was one of the most widely established characteristics of cancer cells, and has been detected in breast, prostate, and different types of brain tumors. Their data suggested that phenotypic changes in phospholipids probably commenced early in carcinogenesis and may be regulated by interplay of cellular immortalization and oncogene transformation. Iorio et al. (2005) confirmed these findings for epithelial ovarian cancer cells reporting abnormal phospholipid metabolism as a common feature in breast and prostate cancer cells, particularly during ovary cancer progression. In line with our findings Robinson et al. (2001) observed changes in rat immune

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function with changes in long-chain fatty acid composition, and with alterations in phospholipids in R3230AC mammanry adenocarcinoma in rats. Bansal et al. (2005) described how parasites induce significant changes in lipid parameters, as has been shown both in vitro and in experimental models in vivo. Similar changes in lipid profile occur in patients having active infections with most parasites. Viral infection may alter the lipid machinery of the host cell thereby modifying the phospholipid composition of the infected cell’s membranes. In addition, various bacteria and parasites are known to have quite different membrane lipid compositions as compared to mammalian cells. Finally, knowing which alterations make cells more or less recognizable by the immune system may also be of importance in the design of cellular vaccines (e.g., to stimulate recognition and activation) and cellular therapies (e.g., to escape recognition and rejection). Acknowledgements The author would like to thank Dr. Michael Glasser, Dept. Biochemistry, University of Illinois for his advice in the use of the LM cell line system as well as the method of phospholipid alteration. Finally, I would like to thank Dr. Raymond Kuhn for helpful discussions in the preparations of the data. References Ackerstaff, E., Glunde, K., Bhujwalla, Z.M., 2003. Choline phospholipid metabolism: a target in cancer cells? J. Cell Biochem. 90, 525–533. Alexander, P., Connell, D.I., Mikelska, A.B., 1966. Treatment of a murine leukemia with spleen cells or sera from allogenic mice immunized against the tumor. Cancer Res. 26, 1508–1515. Allavena, R., Introna, M., Mangloni, C., Mantovani, A., 1981. Inhibition of natural killer cell activity by tumor-associated lymphoid cells from ascites ovarian carcinomas. J. Natl. Cancer Inst. 67, 319–329. Alving, C.R., Beck, Z., Karasavva, N., Matyas, G.R., Rao, M., 2006. HIV-1, lipid rafts, and antibodies to liposomes: implications for anti-viral-neutralizing antibodies (Review). Mol. Membr. Biol. 23 (6), 453–465. Ames, G.F., 1968. Lipids of Salmonella typhimurium and Escherichia coli: structure and metabolism. J. Bacteriol. 95, 833–843. Baldarin, R.W., Barker, C.R., 1967. Tumor specific antigenicity of amino-azo-dye induced rat hepatomas. Int. J. Cancer 2, 355–360. Bansal, D., Bhatti, H.S., Sehgal1, R., 2005. Role of cholesterol in parasitic infections. Lipids Health Dis. 4, 1–7. Berke, G., Amos, D.B., 1973. Cytotoxic lymphocytes in the absence of detectable antibody. Nat. (Lond.) New Biol. 242, 237–239. Berke, G., Tzur, R., Inbar, M., 1978. Changes in fluorescence polarization of a membrane probe during lymphocyte-target cell interaction. J. Immunol. 120, 1378–1384. Berke, G., McVey, E., Hu, V., Clark, W.R., 1981. T lymphocyte-mediated lympholysis. II. The role of target cell histocompatibility antigens in recognition and lysis. J. Immunol. 127, 782–787. Berke, G., Hu, V., McVey, E., Clark, W.R., 1986. T lymphocyte-mediated lympholysis. I. A common mechanism for target recognition in specific and lectin-dependent cytolysis. J. Immunol. 127, 776–781. Blank, K.J., Freedman, H.A., Lilly, F., 1976. T-lymphocyte response to Friend virusinduced tumor cell lines in mice of strains congenic at H-2. Nature 260, 250–252. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Engers, H.D., Unanue, E.R., 1974. Antigen-binding thymic lymphocytes: specific binding of soluble antigen molecules and quantitation of surface receptor sites. J. Immunol. 112, 293–304.

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