In vitro induction of primary and secondary xenoimmune responses by liposomes containing human colon tumor cell antigens

CELLULAR IMMUNOLOGY 71,224-240 (1982)

In Vitro Induction of Primary and Secondary Xenoimmune Responses by Liposomes Containing Human Colon

Tumor Cell Antigens’

LEONARD RAPHAEL AND BALDWIN H. TOME

Departments of Surgery and of Biochemistry and Molecular Biology, The University of Texas Medical School, MSMB 6240, 6431 Fannin, Houston, Texas 77030

Received January 27, 1982; accepted May 31, 1982

Liposomes prepared with human LS 174T colon tumor cell membranes induce specific primary and secondary xenogeneic immune responses in BALB/c splenocytes in vitro. The multilamellar vesicular liposomes were prepared by adding sonicated membrane fragments in 8 mM CaClr to a dried lipid film. Cytotoxic splenocytes generated in vivo exhibited specificity for the LSl74T celh liposomes elicited higher levels of cytotoxicity than did membranes (P < 0.01). Secondary blastogenic responses elicited in in viva-primed spleen cells by liposome- antigens also produced a significantly greater (P i 0.005) response than membranes. Subse- quently, in vitro induction of primary blastogenic and cytotoxic responses by liposome-antigens were accomplished and revealed similar kinetics to that of whole LSl74T cell immunogens. Specificity of the in vitro-primed spleen cells was clearly demonstrated (P < 0.0 1) on a variety of human tumor cells using both the primed lymphocyte and cell-mediated cytotoxicity assays. The results of competitive inhibition tests with autologous lymphoblasts demonstrated that 30% of the cytotoxic activity was directed against lymphocyte antigens. Incorporation of tumor antigens into liposomes has thus enabled primary immunization in vitro to human colon cancer antigens and may afford an adaptable means to evaluate and to select desired immune responses, as well as to identify colon tumor-specific determinants.

INTRODUCTION

Early experimental and clinical evidence (1, 2) has shown that lymphocytes provide a major defense against neoplasia. In order to investigate the mechanisms of lymphocyte-tumor cell interactions and to apply this information to specific cancer immunotherapy, development of a convenient and reproducible assay system is necessary. Due to the complexity inherent in in viva studies our focus has been on developing an in vitro system in which immune recognition of the relevant determinants on the tumor cell could be evaluated. In previous in vitro studies by Sharma et al. (3) and Schecter et al. (4), in vitro sensitization of human lymphocytes to whole tumor cells has yielded only low levels of cytotoxic lymphocytes (CTL).3

’ Supported by NIH Bredoctoral Training Grant, GM07542, NCI/DHHS Grants CA24024, and Re- search Career Development Award 5-K04 CA00579 (B.H.T.).

* To whom correspondence and reprint requests should be sent. 3 Abbreviations used: HBSS, Hank’s balanced salt solution; MEMlO, minimum essential medium

supplemented with 10% fetal calf serum; MMC, mitomycin C, PC, phosphatidylcholine; C, cholesterol;

224

0008-8749/82/120224-17$02.00/O Cqyrighl 8 1982 by Academic Press. Inc. AI1 right8 of reproduction in any farm reserved

INDUCTION OF IMMUNE RESPONSES BY LIPOSOMES 225

Attempts to utilize subcellular moieties to generate cytotoxic lymphocytes have also been marginally effective. For example, Corley et al. (5) showed in human studies that subcellular fractions of lymphoblasts were capable of stimulating weak proliferative and weak cytotoxic responses. In murine studies, Lemonnier et al. (6) demonstrated that while P8 15 murine mastocytoma membranes could elicit a strong secondary cytotoxic response, only a weak primary response was induced. The determination of the specific antigens and associated factors necessary for immune induction in the above studies would be very difficult and probably impossible, due to the complexities (heterogeneity) of the antigenic stimuli.

Recently, with the development of the lipid vesicle (liposome) as an antigen carrier (see Tom and Six (7)) a powerful approach has become available to systematically evaluate distinct antigenic moieties. For example, Hale et al. (8) were able to induce secondary anti-Sendai CTLs using liposomes composed of hemagglutinin-neur- aminidase glycoproteins of Sendai virus and the H-2Kk glycoprotein. A more recent study by Herrmann et al. (9) showed that the inclusion of a detergent-insoluble membrane matrix into liposomes containing purified H-2Kk enhanced the stimulation of secondary CTLs over liposomes without the matrix. In addition Hale et al. (10) reported that liposomes bearing viral and H-2 antigens generated primary cytotoxic T lymphocytes, but only in the presence of concanavalin A supernates. Concanavalin A supernates were also used by Humphries et al. ( 11) to aid induction of in vitro plaque-forming cells to trinitrophenylhaptenated liposomes.

Our investigations have focused on the use of liposomes to generate primary in vitro xenoimmune responses in mouse splenocytes to human colon tumor antigens. The use of murine xenoimmune systems to study HLA antigens is well documented and indicates that the cell-mediated immune mechanism controlling both the rec- ognitive and destructive phases of T-cell-mediated cytotoxic reactions are similar for allogeneic and xenogeneic antigens ( 12-14). Indeed, liposomes containing purified HLA-A and HLA-B, and HLA-DR molecules have been successfully used by Englehard et al. ( 15) and Burakoff et al. ( 16) respectively, to elicit xenogeneic mouse cytotoxic T lymphocytes against HLA antigens by culturing them with in vivo human lymphocyte immunized mouse spleen cells. Furthermore, xenoimmune systems have recently been employed to study cell-mediated mechanisms in tumor immunity of different species by Mathisen and Rosenberg ( 17) and by us (18, 19). The present report describes studies demonstrating that liposomes are indeed able to not only induce secondary, but also primary cytotoxic murine lymphocytes against human colon tumor antigens by in vitro culture.

MATERIALS AND METHODS

Target cells. Human tissue culture cell lines were maintained in MEMlO, i.e., minimum essential media (MEM) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 &ml streptomycin, 2 mi%f glutamine, and 20 mM Hepes buffer, pH 7.2 (details in (20, 21)); these included LS174T, SW1083, COLO 205 (all colon carcinomas), SW780 bladder carcinoma, SW962 vulva carcinoma, and

PA, phosphatidic acid; MLV, multilamellar vesicles; CTAB, 4% cetyl trimethylammonium bromide (citrimide); MLTI, mixed lymphocyte (splenocyte)-tumor cell interaction; CTL, cytotoxic lymphocytes; Hepes, 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid; SD, serologically defined.

226 RAPHAEL AND TOM

MB436 breast carcinoma. The lymphoblastoid cell line COLO 462V, derived from the very patient providing LS174T cells, was maintained in McCoy’s 5a medium, with 0.1 mh4 B-mercaptoethanol and supplemented with 10% fetal calf serum. Monolayer target cells were trypsinized, washed, and labeled for 5-6 hr with 100 &i of Na5’ Cr04 (Amersham Corp, 3.2 &i Cr/ml) per 1 X 10’ cells/ml in MEMlO, at 37°C with 5% COz. These cells were treated with mitomycin C prior to use by incubating 5- 10 X 1 O6 cells/ml with 50 pg/ml of mitomycin C (MMC, Sigma Chem- ical Co., St. Louis, MO.). The labeled target cells were washed three times with HBSS, resuspended in MEM 10, and plated into flat-bottom well tissue culture plates (Falcon Microtest II) at lo4 target cells per well and incubated overnight at 37°C in 5% C02.

Immunization. One to two-month-old female BALB/c mice (Timco, Texas) were injected intraperitoneally with 2 X 10’ LS 174T colon tumor cells, membrane vesicles alone ( 100 pg protein) or membrane vesicles in liposomes (100 pg protein). Spleens were harvested from the animals 7 days after immunization.

Preparation of spleen cells. Mice were killed by cervical dislocation. The spleens were removed and collected in a sterile disposable 60-mm petri dish (Falcon Plastics, Los Angeles, Calif.) containing 5-10 ml of Hank’s balanced salt solution, pH 7.2 (HBSS). The spleen cells were prepared by gently expressing minced spleen pieces in HBSS through a 60-mesh stainless-steel screen with a rubber policeman. The splenocytes were collected by centrifugation at SOOg for 10 min, resuspended in 0.14 it4 NH,Cl and 17 ti Tris, pH 7.4, for 2 min to lyse erythrocytes, and then centrifuged for 9 min. The cells were washed three times with HBSS and resuspended in MEMlO.

Treatment of spleen cells with anti-8 serum plus complement. Affinity-purified, monoclonal antiserum to the 8 alloantigen of SJL mice (anti-Thy 1.2), obtained from clone 30-H 12 (22) was purchased from Becton-Dickinson (Mountain View, Calif.). Spleen cells at 2 X 106/ml in MEM were incubated at a 1: 100 dilution of the purified monoclonal antibody or normal serum from BALB/c mice for 30 min at 4°C. Rabbit complement (Low-Tox-M Rabbit Complement, Cedarlane Labo- ratories, London, Canada) was then added at a dilution of 1:20, and incubation continued at 37°C for an additional 30 min. The cells were incubated with 0.1% trypan blue to determine their viability.

Membrane isolation. Membranes were isolated by a modification of the method of Brunette and Till (23). Twenty-four hours before beginning the procedure, 8 X 10’ LS 174T tumor cells were trypsinized, washed, suspended in 500 ml MEM, and cultured in a spinner flask. At the end of 24 hr, the cells were removed from the spinner flask and washed twice with cold phosphate-buffered saline, pH 7.2 (PBS). The cells were then resuspended in 25-ml aliquots of water supplemented in 1 mM each of calcium, magnesium, and phenylmethylsulfonyl fluoride for 45 min. No more than 5 X 10’ cells were contained in each 25-ml aliquot. Each aliquot was then treated in a Dounce homogenizer with a tight-fitting pestle at a speed of 5 and given 35 strokes. The suspension was periodically examined microscopically for cell breakage. The lysate was spun down in a Sorval RC-5B Superspeed Cen- trifuge for 20 min at 12,000g. At this point the supematant fluid was discarded and the pellet was resuspended in a two-phase polymer (20% Dextran T-500 and 30% PEG 1000 with sodium phosphate at a pH of 6.6). This preparation was spun at 14,OOOg for 10 min at 4°C and the membranes were removed from the interface


formed by the two polymers; the cell pellet was resuspended and reprocessed to increase the membrane yield. The harvested membranes were pooled, washed, and resuspended in phosphate-buffered saline, and frozen at -70°C.

Liposome preparation. Multilamellar vesicles (MLV) were prepared by a modification of the method described by Bangham et al. (24). The harvested plasma membranes bearing colon cancer antigens were sonicated at 35 W for 1 min in a cuphorn at setting 2 (Heat Systems, Inc., Plainview, N.Y.); these membrane vesicles were pelleted by centrifugation (2O,OOOg, 45 min) and washed once in PBS; 2 mg (evaluated by Lowry protein) of sonicated membranes were added to a dried lipid film (total lipid ranged from 65 to 83 pg) of 7:2: 1 molar ratios of phosphatidylcholine (PC), cholesterol (C), and phosphatidic acid (PA) with subsequent addition of 0.5 ml 8 rUf CaC12. A magnetic stirring bar was added to this mixture and stirred for 12 hr at 37°C under N2 gas. The liposomes were pelleted and washed three times with PBS (10,OOOg 30 min) to eliminate unincorporated membrane vesicles which did not pellet until 20,OOOg. A protein determination by a modified Lowry assay (25) and a phosphorus assay (to standardize phospholipid concentrations) by the Bartlett technique (26) were performed on each final preparation.

For the in vitro immunizations, the maximal amount of liposomal lipid which did not significantly affect the blastogenic response of immune spleen cells was determined (data not presented). Accordingly, 100 pg or less of phospholipid in the form of liposomes did not significantly alter the primary and secondary blastogenic responses of spleen cells to LS 174T. Furthermore, 80 pg or less of phospholipid did not affect the cytotoxic capacity of primed spleen cells. Thus, the amounts of liposomes used in the in vitro cultures were kept within these levels.

Blastogenesis of immunized cells. Responding spleen cells (2 X 106) obtained 5 days after in vitro tumor antigen-splenocyte cultures or in vivo from mice immunized 7 days earlier with an intraperitoneal injection of tumor cells, membranes, or liposome-antigens were added to the antigens presented by MLV-membrane (25 rg), membranes alone (25 rg), or MMC-treated LS174T tumor cells (50: 1, responder:stimulator) in a total volume of 200 ~1 in wells of flat-bottom Microtest II plates and incubated for l-5 days at 37°C in 5% COZ and humidified air. Cultures were pulsed for 24 hr with 1 &i per well of [3H]thymidine, New England Nuclear, 1 Ci/ml) before harvesting on fiberglass filter paper with a vacuum havester (Mi- crobiological Associates). The filter disks were treated in 5 ml of spectrafluor scin- tillation fluid (Amersham Corp., Arlington Heights, Ill.) and counted in an LS8 100 beta counter (Beckman Instrument, Irvine, Calif.).

Primed lymphocyte assay of in vitro-primed cells. Spleen cells from normal mice ( 100 X lo6 in MEM 10) were coincubated in a T75 tissue culture flask (Falcon Plas- tics, Oxnard, Calif.) at 37°C 5% CO1 with (i) 2 X lo6 mitomycin C-treated stimulator cells at 5O:l responder-to-stimulator ratio, (ii) membrane vesicles with 100 pg protein, or (iii) liposomes with 100 pg membrane protein. On Days 3, 5, and 7, 5 ml of fresh MEM 10 was added to each flask. On Day 10, the spleen cells were removed and washed in HBSS and 5 X lo5 viable, primed spleen cells in 100 ~1 were incubated in microtiter wells with 100 ~1 of 2 X lo4 mitomycin C-treated stimulator cells. On Day 11, the cultures were pulsed (for 24 hr) with 1 &i per well of [3H]thymidine and vacuum harvested on Day 12. The amount of antigen (100 pg) presented to the lymphoid cells in the in vitro sensitization by the membrane and liposome preparations was estimated (by Lowry proteins) to be four times the

228 RAPHAEL AND TOM

amount available in the membranes presented by 2 X 10” whole tumor cells. An- tigen-free “empty” liposomes as used in these experiments did not generate blastogenic or cytotoxic spleen cells in any of the assays.

Preparation of mixed-thymocyte culture-conditioned medium. A sterile single cell suspension from an equal number of BALB/c and C3H/HeJ mouse thymus glands in MEM 10 was prepared. The thymocytes were cocultured at 2-4 X lo6 cells/ml (50 ml/75 cm* tissue culture flask) for 48 hr in 5% CO2 at 37°C. This thymus cell suspension was harvested and centrifuged for 10 min at 300g. Supematant fluids were decanted, passed through 0.2-pm filters, and stored in lo-ml aliquots at -70°C. This conditioned media was used in experiments at a 25% concentration.

Generation of cytotoxic activity. Spleen cells (100 X lo6 in MEM 10) from normal (i.e., in vitro primary immunization) or immune mice (i.e., in vitro secondary stimulation) were cultured at 37°C 5% CO2 in a T75 flask with stimulator cells (50: l), membranes ( 100 pg), or liposome-antigens ( 100 pg protein) in MEM 10 and 5 X 10m5 M P-mercaptoethanol. On Day 3, 1 ml of nutritional mix containing MEM, Eagle’s with nonessential amino acids, essential amino acids, vitamins, and NaHC03 (27) was added to each flask. In addition, a 10% CO*, 7% O2 gas mixture was bubbled into the flasks for 20 set every 2 days. This gas mixture of Mishell and Dutton (27) did not alter blastogenic results but appeared to enhance subsequent cytotoxicity results. On Day 5, the cultured splenocytes were removed, washed, counted, and tested for tumor cell cytotoxicity. For the dose-response studies, normal spleen cells were cultured, as described above, with increasing amounts of liposomes (0. l- 1000 fig protein) and on Day 5, the cultured splenocytes were tested for cytotoxicity on LS 174T targets.

Competitive inhibition of target cell lines by the addition of nonlabeled cells. The assay for cell-mediated “Cr release was performed as above with the exception that effector spleen cells were incubated in wells with nonlabeled target cells and labeled target cells. The ratios of effector:cold inhibitor cells tested were 10: 1, 20: 1, and 50: 1. Each reaction mixture had a final volume of 0.2 ml, consisting of 0.1 ml (2.0 X 106) of spleen cells, 0.05 ml of competing cells, and 0.05 ml (1 X 104) of labeled target cells.

Cell-mediated cytotoxic assay. Effector spleen cells (1 X 106) from in vitro or in vivo immunization were used at a 1OO:l effector-to-target cell ratio. The effecters and targets in a total volume of 200 ~1 were incubated for 18 hr at 37°C 5% CO*. After incubation the plates were centrifuged at 200g for 4 min and 100 ~1 of media removed from each well for counting in a Gamma 4000 counter (Beckman Instru- ments).

Evaluation of data, Percentage observed 51Cr release (cytotoxicity) was calculated as 100 X (E - S/CTAB-S), where E is the fraction of “Cr released by antigen-stimulated effector cells, S is the fraction of 5’Cr released from labeled target cells incubated with media alone, and CTAB is the maximum release of 51Cr by 4% citrimide.

Specifity of the cytotoxic response was calculated using two-way analysis of variance (28) as described for cytotoxicity data by Takasugi and Mickey (29). The interaction analysis allows one to statistically set a baseline for cytotoxicity assays that accounts for variability in target susceptibility and the differential potency of effector cells. The specific cytotoxicity represents the deviation of each observed cytotoxicity from the experimental baseline caused solely by specific interaction


between target and effector cells. Test results are displayed as a two-way table in which rows correspond to effector cells and columns correspond to target cells.

The method of analysis allows quantitative comparison of results across many target cells with different sensitivities. It evaluates not only the effect of one splenocyte suspension on several different tumor targets, but also the effect of different splenocyte samples on each target cell. By computing average reactivity and sen- sitivity, it predicts the expected nonspecific cytotoxic activity that must be surpassed by the total reaction for specific cytotoxicity to be identified. Confidence intervals at the 5% significance level were constructed from the t distribution and the calculated variance for each experiment.

Significance between results for the blastogenic assays was established when P < 0.05 as computed with Student’s t test.

RESULTS

In Vivo-Primed BALB/c Splenocytes

Blastogenesis. The ability of antigen-bearing liposomes to stimulate secondary blastogenic activity from in vivo LS174T-primed cells was compared with intact LS174T cell and membrane immunogens by in vitro culture (Table 1). It can be seen that all three immunogens used as secondary stimulators elicited significantly higher blastogenic responses than immune splenocytes cultured in the absence of exogenous antigens. Incubation of unprimed splenocytes with these three preparations resulted in stimulation of [3H]thymidine uptake but the levels were lower than those seen in immunized splenocytes from secondary stimulation (data not shown). (Note that the latter control is essentially that set up in the in vitro primary immunization protocol described below and the focus of this paper). The levels of [3H]thymidine uptake induced were generally lower for immune cells cultured with membranes or liposomes than for spleen cells cultured with whole cells. More im-

TABLE 1

Secondary Blastogenic Response of Immune BALB/c Spleen Cells to LS 174T Cells, Membranes, and Liposomes

Experiment Normal

splenocytes None

Secondary stimulator ’

LS 174T Cells* LS 174T Membrane* Liposomes”’

1 7151 + 1263d 8255 f 3751 36,546 f 2689 19,316 f 5068 26,992 + 4505 2 7165 zk 624 7962 + 2215 15,782 + 2591 11,335 It 599 21,732 + 2856 3 5915 + 1405 7481 + 795 26,393 of: 2703 13,443 + 1529 21,044 + 5118

’ Mouse splenocytes from LS174T immune animals were cocultured with the secondary stimulators at a responder-to-stimulator ratio of 5O:l. For secondary stimulation, 50 pg protein (26 pg lipid) of LS174T membrane and 50 rg protein (28 pg lipid) of liposomes were used. After 4 days in culture, [‘Hlthymidine was added and the cells were harvested on a MASH.

* Experimental group exhibits significantly greater blastogenesis than immune spleen cells with no stimulator, P < 0.00 1.

’ Liposome group exhibits significantly greater reactivity than membrane stimulated group in all three experiments, P < 0.0 1.

d Counts per min + SD.

230 RAPHAEL AND TOM

portantly, however, liposome-antigens were capable of eliciting a greater response than comparable amounts of membrane-borne antigen.

Speczjic cytotoxicity. The in vivo induction of cytotoxic splenocytes was examined after immunization of mice with LS 174T cells, membranes, and liposomes. Immune splenocytes were cocultured in vitro with either the sensitizing antigen or without antigenic stimulators for 5 days. The elicitation of specific cell-mediated cytotoxicity was demonstrated using a panel of human tumor cell targets. Specificity was evaluated using inteiaction analysis. Five sets of identical experiments were run. Ob- served, as well as computed, specific cytotoxicity results from a representative experiment are presented in Table 2. The data presented as “observed cytotoxicity” and calculated in the conventional manner reflect an apparent nonspecific target cell destruction. This can be expected if different targets have varying sensitivities to lysis. This is especially true in heterogenous human target cell combinations. Utilizing interaction analysis, it can be seen that immune spleen cells cocultured with the original sensitizing antigen (LS174T cells or liposomes) displayed specific cytotoxicity to LS 174T while the immune spleen cells cultured alone without any stimulating antigen source showed no reactivity to LS174T targets. However, the group immunized with membranes exhibited reactivity for COLO 462V, the lymphoblast counterpart to LS174T. Spleen cells cocultured with SW780, and MB436, respectively, demonstrated specific cytotoxicity against their labeled targets. Al- though we are convinced that this interaction analysis of Takasugi and Mickey (29) is more reflective of the “true” cellular specificity, we have included the conventional evaluation for comparison. As in the blastogenic results, liposome-antigens were capable of eliciting stronger cytolytic responses than membrane antigens with equiv- alent amounts of protein.

In Vitro-Primed BALB/c Splenocytes

Blastogenesis of mixed splenocyte-tumor cell interaction (MLTI). The in vitro kinetics of blast formation in a primary mixed splenocyte-tumor cell interaction (MLTI) of spleen cells cultured with MMC-treated LS174T cells, membranes, or liposomes are illustrated in Fig. 1. Four similar experiments were run. It can be seen that while LS 174T cells and liposomes elicit significant proliferative responses, membranes produce very little reactivity. The kinetics of cellular proliferation seen here is similar to that shown by Enger in an allogeneic system (30).

The specificity of a primary blastogenic response generated in vitro was tested with different tumor cell stimulators in a blastogenic restimulation of in vitro sensitized spleen cell at 10 days. The results in Fig. 2 illustrates that effector cells generated in vitro with intact cells or liposomes responded strongly when cultured with colon tumor cells and weakly with other target cell types. Normal splenocytes cultured alone for 10 days demonstrated no reactivity against any of the secondary stimulators. Again, liposomes were more effective than membranes in restimulating immune (memory) cells to colon tumor antigens.

Cytotoxicity. The kinetics of cell-mediated cytotoxicity by the spleen cells from in vitro sensitization are shown in Fig. 3. This result is representative of three identical experiments. The development of cytotoxicity generally followed the pattern of cellular proliferation seen in Fig. 1, in that the extent of cytotoxicity corresponded to the demonstration of blastogenic activity. The peak of activity for all sensitized

TABL

E 2

Spec

ific

Cyt

otox

icity

of

in

Viva

-Prim

ed

BALB

/c

Sple

nocy

tes

Effe

ctor

cel

l

Targ

et

cell

Cyt

otox

icity

LS c

ell

imm

une

+ LS

174T

Mem

bran

e im

mun

e +

mem

bran

es

Lipo

som

e im

mun

e +

lipos

omes

SW78

0 im

mun

e +

SW78

0

MB4

36

imm

une

+ M

B436

LS c

ell

2 im

mun

e Ta

rget

al

one

z su

scep

tibilit

y ,+

ij

LS I7

4T

Obs

erve

d +4

3.7”

+3

4.0

+57.

8 10

.9

+24.

8 -2

2.8

24.7

2

Spec

ific

12Sb

4.

9 21

.4

-12.

0 -0

.6

-25.

9 8 s

COLO

46

2V

Obs

erve

d 14

.7

17.8

13

.9

10.6

11

.6

-35.

2 5.

6 Sp

ecifi

c 2.

6 7.

8 -3

.4

6.8

5.3

-19.

2 B 2

SW78

0 O

bser

ved

11.3

12

.0

21.0

+4

1.7

18.4

-1

5.4

14.8

Sp

ecifi

c F

- 10

.0

-7.2

-5

.5

28.7

2.

9 -8

.6

c1

SW96

2 O

bser

ved

-27.

7 -3

3.2

-25.

2 -4

9.7

-46.

1 -2

1.9

34.0

H

Spec

ific

-0.2

-3

.6

-2.9

-1

3.9

-12.

8 33

.7

8

MB4

36

SW10

83

Obs

erve

d Sp

ecifi

c

Obs

erve

d

Spec

ific

Effe

ctor

pot

ency

14.8

15

.6

13.1

-1

0.3

+24.

8 -1

3.1

7.5

z

0.8

3.7

-6.1

-1

6.0

16.6

1.

0 I=

14.0

t5

12

.2

21.4

17

.6

2.4

+10.

7 13

.1

-5.6

-5

.3

-3.4

6.

3 -1

1.4

19.2

i! In

11

.8

9.7

17.0

3.

5 6.

0 -1

6.3

5.3

in

Not

e. C

onfid

ence

in

terv

al

at t

he 0

.05

conf

iden

ce

leve

l is

k5.

21

for

effe

ctor

-targ

et

inte

ract

ions

; fo

r co

mpa

rison

s be

twee

n ef

fect

or c

ell

pote

ncie

s f3

.54;

an

d fo

r co

mpa

rison

s be

twee

n ta

rget

cel

l su

scep

tibilit

ies

+ 3.

54.

’ The

hi

ghes

t “o

bser

ved

cyto

toxi

city

” fo

r ea

ch e

ffect

or c

ell

grou

p is

den

oted

wi

th

plus

sig

n pr

eced

ing

the

resu

lt.

b The

hi

ghes

t co

mpu

ted

“spe

cific

cy

toto

xici

ty”

for

each

effe

ctor

cel

l gr

oup

is u

nder

lined

.

- -

-.

232 RAPHAEL AND TOM

30-

24-

18-

12-

6-

lmmunagen lmmunagen

’ None ’ None 0 LS1741 Cells 0 LS1741 Cells

aLS174TMembranes aLS174TMembranes

n LS1747 L~poromes n LS1747 L~poromes

Time (days)

FIG. 1. Kinetics of primary mixed splenocyte-tumor cell interaction. BALB/c splenocytes (100 X 106) from normal animals were cocultured with no stimulator, MMC-treated LS 174T cells at a responder-to- stimulator ratio of 50~1, LSl74T membranes (100 pg protein, 65 fig lipid), or liposome-antigens (100 pg protein, 75 fig lipid). Blastogenesis assayed by [3H]thymidine incorporation (mean k SEM) was determined for Days 2, 3, 4, and 5.

spleen cells was Day 5. The low levels of cytotoxicity generated with membranes corresponded to the lack of proliferation seen in Fig. 1. However, the greater proliferation induced by liposomes did not correlate with the production of CTLs, as intact cells elicited greater killing.

The effect of varying the concentration of liposome-antigens on the appearance of lytic activity in primary cultures of spleen cells is shown in Fig. 4. A dose-response

Orearl (Cd0 205) (MB 436)

Secondary Stimulator

Bladdsr (SW 780

FIG. 2. Secondary blastogenic response of in vitro colon tumor antigen-primed BALB/c splenocytes. Mouse splenocytes (100 X 106) from normal animals were cocultured witb no stimulator, MMC-treated LSl74T cells, LSl74T membranes (100 pg protein, 58 a lipid), or liposomes (100 pg protein, 70 fig lipid) and assayed for blastogenesis by [3H]tbymidine incorporation (mean f SEM) on Day 12.

INDUCTION OF IMMUNE RESPONSES BY LIF’OSOMES 233

24-

lmmunogen

l None

0 LSl741 ceils A LS174TMembrancr n LS174TLiposom~s

Time (days)

FIG. 3. Kinetics of in vitro generation of cytotoxic T lymphocytes. BALB/c splenocytes (100 X 106) from normal animals were cocultured with no stimulator, MMC-treated LSl74T cells at a tesponder-to- stimulator ratio of 50: 1, LS174T membrane (90 pg protein, 47 pg lipid), or liposomes (90 pg protein, 54 pg lipid) at a responder-to-stimulator ratio of 50: 1. Cytotoxicity assayed by ” Cr release was determined for Days 2, 3, 4, 5, 6, and 7.

relationship was observed up to 100 pg protein. At this concentration of liposomes, 30% cytotoxicity on labeled LS 174T cells was obtained. Cytotoxic cells were elicited with as low as 10 pg protein.

The specificity of the cytotoxic response generated in vitro was determined (re- peated in seven experiments) after culturing spleen cells from normal mice with MMC-treated intact cells, membranes, or l&some-antigens for 5 days and subse- quently testing for cytotoxicity on a panel of human target cells. The results are presented in Table 3. Normal splenocytes, immunized with LSl74T cells, membranes, and liposome-antigens developed cytotoxicity against LS 174T, but only in-

Lipmoma -Born4 Protein (up)

FIG. 4. Dose responses for cytotoxic splenocyte generation by liposomes. BALB/c splenocytes (100 X 106) from normal animals were cocultured with increasing amounts of LS174T liposomes (protein). Cytotoxicity assayed by “Cr release was determined after 5 days. Shaded area represents the mean + SD of the negative control (i.e., target cell lysis in the presence of normal spleen cells).

TABL

E 3

Spec

ific

Cyt

otox

icity

of

in

Vitro

-Prim

ed

BALB

/c

Sple

nocy

tes

Effe

ctor

cel

l

Targ

et c

ell

Cyt

otox

icity

N

orm

al

(N)

N+

LS c

ells

N+

M

embr

anes

N+

Li

poso

mes

N+

SW

1083

N+

sw

180

Targ

et

susc

eptib

ility

LSl74

T O

bser

ved

-3.8

+2

5.5”

5.

5 9.

9 -4

.0

-4.4

4.

8 Sp

ecifi

c -1

5.9

17.9

6 0.

2 11

.8

-1.9

-1

2.0

COLO

46

2CV

Obs

erve

d 15

.2

-8.2

-3

.2

-25.

4 -1

5.1

fl4.4

-3

.1

Spec

ific

11.6

-1

.3

0 15

.0

-4.5

15

.3

$ SW

780

Obs

erve

d -2

5.8

24.5

-2

3.9

-22.

9 -1

8.9

5.1

18.5

i2

Sp

ecifi

c -1

4.6

-8.8

-5

.9

2.3

6.5

20.8

Ei

SW96

2 O

bser

ved

22.1

-5

.0

-13.

9 -2

4.3

-27.

8 -1

4.5

-10.

6 Sp

ecifi

c 25

.4

2.8

-3.8

-7

.0

-10.

3 -6

.7

i M

B436

O

bser

ved

+34.

3 11

.3

+11.

4 8.

6 2.

5 11

.2

13.2

Sp

ecifi

c 13

.9

-4.1

-2

.3

2.1

-3.8

-4

.8

HT29

O

bser

ved

6.2

5.9

11.0

-7

.6

-6.0

1.

8 -1

.8

Spec

ific

-11.

7 4.

9 12

.3

.9

2.1

-8.8

SW 1

083

Obs

erve

d 2.

7 1.

8 3.

7 0.

8 +8

.5

3.2

3.6

Spec

ific

-8.2

-4

.6

-.4

3.4

11.8

-3

.2

Effe

ctor

pot

ency

5.

5 1.

0 -1

.3

-8.5

-8

.7

1.0

-1.8

Not

e. C

onfid

ence

in

terv

al

at t

he 0

.05

conf

iden

ce

leve

l is

k4.

07

for

effe

ctor

-targ

et

inte

ract

ions

; fo

r co

mpa

rison

s be

twee

n ef

fect

or c

ell

pote

ncie

s e2

.86;

an

d fo

r co

mpa

rison

s be

twee

n ta

rget

cel

l su

scep

tibilit

ies

k2.6

5.

’ The

hi

ghes

t “o

bser

ved

cyto

toxi

city

” fo

r ea

ch e

ffect

or c

ell

grou

p is

den

oted

wi

th

plus

sig

n pr

eced

ing

the

resu

lt.

b The

hi

ghes

t co

mpu

ted

“spe

cific

cyt

otox

icity

” fo

r ea

ch e

ffect

or c

ell

grou

p is

und

erlin

ed.


tact cells and liposomes were capable of inducing significant levels of specific cytotoxicity. As in the in vivo results (Table 2) cross-reactivities are exhibited. Normal spleen cells when grown alone for 5 days showed no specific cytotoxicity to LSl74T, but they did show significant levels of natural cytotoxicity for SW962 and MB436. As a control for the statistical evaluation, in vitro sensitization of naive spleen cells with SW 1083 and SW780 cells was carried out and these also generated effector cells with maximal specific reactivities toward the homologous target antigens. Thus, this ability to sensitize against other homotypic and heterotypic histologic cell types indicates that the LS 174T cells are not unique in their immunogenic capacity.

As a means to further evaluate the specificity, competitive inhibition tests were performed with in vitro sensitized spleen cells. Results of this experiment are depicted in Fig. 5. As expected, the addition of nonlabeled LS174T cells to the reaction mixture was found to reduce the cell-mediated cytotoxicity of “Cr-labeled LS 174T cells to background levels. These results also show that the lymphoblastoid line COLO 462V from the very patient providing the colon tumor cells, LS 174T, caused a significant inhibition (30%) of cell-mediated “Cr release from LS174T cells at 20: 1 and 10: I effector:inhibitor ratios. In contrast the addition of unlabeled MB436, SW780, and SW1083 caused no inhibition of cy-totoxicity.

The effect of thymocyte-conditioned media on the generation of CTLs in the in vitro immunization is shown in Table 4. Clearly the addition of conditioned media resulted in an improvement in the ability of the antigens to elicit anti-LS174T effector cells, especially in the observed cytotoxicities. In fact, in the absence of the conditioned media, membranes were not immunogenic (compared with Table 3). There is also a complete correspondence between the observed and computed specific cytotoxicities for all six effector cell-target cell pairs. Similar results were found in five additional experiments.

As a preliminary characterization of the effector cells generated in vitro by the liposome-antigens, cell populations obtained after 5 days of culture were treated with anti-Thy 1.2 serum and complement before assaying their lytic activity on the relevant target cells. The residual cells were not reconstituted with additional cells in the assay. As shown in Table 4, this treatment abolished the lytic activity of the LS 174T immune cells as seen in the specific cytotoxicity results indicating that the effector cells generated in the primary in vitro cultures were T lymphocytes, or required T-lymphocyte function.

DISCUSSION

The results reported here are the first to demonstrate that human colon tumor antigens incorporated into phospholipid vesicles are capable of stimulating specific primary and secondary cytotoxic responses in vitro. The ability to incorporate surface antigens into liposomes and elicit primary immune responses thus complement and extend the observations that human histocompatibility antigens on liposomes are capable of eliciting secondary cytotoxic lymphocytes in a xenogeneic system (8, 9). In addition, the liposome-borne antigen preparations in our studies were capable of inducing comparable levels of cytotoxicity to those generated in the murine system of Hale et al. (10).

The observation that liposome-antigens induced specific cytotoxic T lymphocytes in vivo by Day 7 is consistent with the time course of appearance of cytotoxic T

236 RAPHAEL AND TOM

MB436 (6roaot) SW7ETt (BhddarJ SW1083 (Colon)

COLO462V (Lymphoblast)

Ettactor : Inhibitor Ratio

FIG. 5. Cold target inhibition of BALB/c spleen cells immune to LS 174T colon tumor cells. Immune spleen cells were first mixed with varying numbers of unlabeled tumor cells followed by testing on %Zr labeled LSl74T cells. Shaded area at the top represents the mean + SD of the positive control (i.e., without inhibitor cells) and at the bottom, the mean f SD of the negative control (i.e., target cell lysis in the presence of normal spleen cells).

lymphocytes (CTL) following allogeneic stimulation (31, 32). The kinetics of the primary blastogenic response of normal spleen cells to intact cells, membranes, and liposomes correspond to the same pattern exhibited by CBA spleen cells when cocultured with allogeneic DBA/2 membrane preparations (33). Peak activity for both interactions occurred on Day 4. Our studies further revealed that the kinetics of the generation of cytotoxic effector cells against LS174T were similar to that found with allograft responses in vitro. The peak cytotoxic response on Day 5 com- pares with the appearance of CTLs to syngeneic tumors (30, 31) and allogeneic tumors (30, 34). Therefore, the xenogeneic cell-mediated immunity generated by liposomes exhibit characteristics found in allogeneic and syngeneic anti-tumor im- munities.

The induction of specific cell-mediated cytotoxicity in vitro was consistently accomplished by cocultivation of normal spleen cells with liposomes. Optimal cytotoxic activity against the colon tumor antigens was generated after 5 days in culture. The pattern of in vitro cytotoxicity (Table 2) paralleled that seen for in vivo immunized cells (Table 3). Whether this observation suggests that similar types of immunity are elicited, or that appropriate and relevant lymphoid cell populations necessary for the generation of in vivo immunity have been retained in the splenocytes for in vitro immunity, remains to be determined.

Liposomes composed of membrane vesicles were effective elicitors of in vitro immune splenocytes. The inability of soluble antigens to induce immune cells may reflect a need for the association of several antigens in a lipid matrix in order to signal the proliferation of necessary cell populations. Additional support for the use of liposome-borne membrane vesicles instead of reconstituted membranes has been reported by Herrmann et al. (11). They found that the stimulating activity of antigen is increased by including a detergent-insoluble membrane matrix fraction during

TABL

E 4

Effe

ct o

f Th

ymoc

yte-

Con

ditio

ned

Med

ia on

in

Vitro

Se

nsitiz

atio

n of

BAL

B/c

Sple

nocy

tes

Effe

ctor

cel

l

N+

Nt

N+

N+

N+

N+

Targ

et

2

Targ

et

cell

Cyt

otox

icity

N

orm

al

(N)

LS C

ells

M

embr

anes

Li

poso

mes

CO

LO46

2V

MB4

36

SW10

83

Thy

1.2”

su

scep

tibilit

y E!

LS17

4T

Obs

erve

d 4.

5 +4

6.1b

+3

4.5

f43.

4 20

.7

11.9

20

.2

+21.

3 25

.3

Spec

ific

-18.

6 16

.0’

10.0

13

.7

-5.1

-9

.3

-8.6

1.

6 $

- -

COLO

462V

g

Obs

erve

d 2.

8 11

.4

19.1

11

.7

+43.

2 5.

1 12

.0

13.1

14

.8

Spec

ific

-9.8

-7

.3

5.1

-7.5

27

.9

-5.6

-6

.3

3.9

- z

MB4

36

Obs

erve

d 8.

4 10

.1

6.8

10.8

2.

9 +2

2.9

6.8

5.8

9.3

2

Spec

ific

1.3

-3.1

-1

.7

-2.9

-6

.9

17.7

-6

.0

2.1

F E

SW10

83

Obs

erve

d +1

0.7

6.0

7.2

2.7

23.0

-2

.0

+36.

2 -2

.6

7.6

Spec

ific

5.3

-5.5

0.

4 -9

.3

-5.8

-5

.5

25.1

-4

.6

$

SW78

0 O

bser

ved

-3.1

-8

.5

-17.

8 -1

.3

-6.5

-1

5.1

-11.

3 20

.3

- 10

.3

E

Spec

ific

9.4

-2.1

-6

.7

4.4

3.3

-0.7

-4

.5

-4.4

2

SW96

2 O

bser

ved

8.8

4.0

-9.2

3.

3 -1

3.8

-1.8

2.

9 -5

.3

- 1.

4 ‘k

Sp

ecifi

c 12

.1

1.5

-7.0

.3

-1

2.9

3.7

.8

1.7

El

Effe

ctor

pot

ency

5.

4 11

.5

6.8

12.0

8.

1 3.

5 11

.1

12.0

7.

6 B

Not

e. C

onfid

ence

in

terv

al

at t

he 0

.05

conf

iden

ce

leve

l is

k3.

62

for

effe

ctor

-targ

et

inte

ract

ions

; fo

r co

mpa

rison

s be

twee

n ef

fect

or c

ell

pote

ncie

s kl

.87;

an

d fo

r co

mpa

rison

s be

twee

n ta

rget

cel

l su

scep

tibilit

ies

k2.4

I.

a M

onoc

lona

l an

tibod

y Th

y 1.

2 pl

us c

ompl

emen

t tre

ated

“N

+ lip

osom

es”

effe

ctor

cel

ls.

b The

hi

ghes

t “o

bser

ved

cyto

toxi

city

” fo

r ea

ch e

ffect

or c

ell

grou

p is

den

oted

wi

th

plus

sig

n pr

eced

ing

the

resu

lt.

’ The

hig

hest

com

pute

d “s

peci

fic

cyto

toxi

city

” fo

r ea

ch e

ffect

or c

ell

grou

p is

und

erlin

ed.

h,

tr:

238 RAPHAEL AND TOM

formation of the liposomes. In this light the efficacy of our liposome preparations may be due to the association of right-side out (exposing surface antigens) and right- side in (exposing cytoplasmic proteins attached on inner surface, e.g., a&n filaments) vesicles on the liposome surface.

A recent report by Hale et al. (12) suggests that the reason subcellular fractions are poor stimulators of primary immune responses is that they lack the ability to stimulate T-cell helper factors necessary for elicitation of cytotoxic T lymphocytes. Thus, the minimal immune response induced by membrane antigens as compared to liposome-antigens may be due to a lack of antigen density or improper antigen orientation necessary for stimulating inductive signals. This is supported by the suggestion of Fast et al. (35) that the size of the subcellular material is critically important in the generation of a CTL response. Our dose-response studies using liposome-antigens reveal that the protein concentration necessary for induction of cytotoxicity may reflect a necessary antigen density on the liposome surface required for immune activation. The addition of concanavalin A or mixed thymocyte culture- derived thymus factors may produce necessary signals for in vitro sensitization with membranes and soluble antigens.

The use of interaction analysis in this study was useful in revealing specific cell- mediated cytotoxic reactions against homologous target cells. When targets with widely varying characteristics were used, the observed cytotoxicities in the system often appeared to produce antispecies directed (nonspecific or cross-reactive) cytotoxicity. For example, note the cytotoxicity of COLO 462V-immunized cells on the target panel in Table 4. When the target cell and the effector cell properties were entered into the computations by interaction analysis, specificities (or lack thereof) to the histologic type antigens become more distinct. It appears that in instances of strong cytotoxic potential of immune cells, both “observed” and “specific” results corresponded (see Tables 3 and 4). On the other hand, when cytotoxicity was weak, the “specific” results computed by interaction analysis, and not the “observed” results, revealed specific reactions on the homologous target cells (Table 3, SW 780 effecters). Based on this interpretation, LS membrane-immune cells in Table 3 are weakly cytotoxic because there was no correspondence between the “observed” and “specific” results. On the other hand, there is correspondence of both computations when thymocyte factors were included in the immunization (Table 4). Accordingly anti-Thy 1.2 treated (liposome-immune) effector cells (Table 4) were not specifically cytotoxic, since there was no correspondence between the two computations. While the use of interaction analysis may be unnecessary in inbred experimental animal systems, it appears that data processing by the latter means was useful in the present studies with human targets.

The cell-mediated component of a xenogeneic response is known to exhibit reactivity to a wide variety of specificities on the sensitizing cell. Several investigators have proposed that the serologically defined (SD) determinants important in allogeneic immunity are also targets for xenogeneic cytotoxic lymphocytes (14, 36). It is unclear whether the antigens being recognized on LS174T are tumor antigens alone, or SD determinants in association with tumor antigens. The observation that autologous lymphoblast cells (COLO 462V) inhibit only 30% of the cytotoxicity in the competitive inhibition studies indicates that SD determinants on the LS174T cell surface may be altered or reduced so that cells immunized to LS174T exhibit minimal anti-lymphocyte antigen activity. The apparent “reduced” expression of


lymphocyte antigens is consistent with the results in Table 4 in which immune COLO 462V effector cells also failed to exhibit reactivity against LS174T cells. We previously described the antigenic markers present on colon tumor cells (37), such as heterophile antigens, normal tissue antigens, organ-specific antigens, and oncofetal antigens. More recently, a colon tumor-asssociated antigen, CSAp, was described by Pant et al. (38). In addition, identification of colon tumor-associated antigens have been reported by Koprowski and colleagues (39,40) using monoclonal antibody probes. Some of the above antigens may indeed be involved as the targets for the specifically cytotoxic lymphocytes. The studies presented herein provide evidence for a colon tumor cell specificity, but do not address themselves to the specific antigenic determinants. Future studies will be necessary to clarify this critical issue- the determination of the exact specificities being recognized by the immune cells and of the role MHC products have in tumor immunity (if any).

In conclusion, the use of liposomes as immunogenic carriers may provide a means to investigate the cell-antigen interactions necessary in the induction of cytotoxic T lymphocytes. Furthermore, the molecular requisites for antigen recognition may be extensively studied through selective incorporation of monoclonal-antibody defined, purified molecules into phospholipid vesicles. Such an approach may allow for the identification of the unique antigenic determinant(s) recognized by the cytotoxic cells. Indeed, these approaches have the potential for application in specific immunotherapy protocols.

REFERENCES

1. Klein, G., Sjogren, H. O., Klein, E., and Hellstron, K. E. Cancer 20, 1561, 1960. 2. Hellstrom, I., Hellstrom, K. E., Evans, C. A., Heppner, G. H., Pierce, G. E., and Yang, J. P. S.,

Proc. Nat. Acad. Sci. (Wash.) 62, 362, 1969. 3. Sharma, B., and Terasaki, P. I., Cancer Rex 34, 115, 1974. 4. Schechter, B., Treves, A. J., and Feldman, M., J. Nat. Cancer Inst. 56(5), 975, 1976. 5. Corley, R. B., Dawson, J. R., and Amos, D. B., Cell Immunol. 16, 92, 1975. 6. Lemonnier, F., Mescher, M., Sherman, L., and Burakoff, S., J. Immunol. 120(4), 1114, 1978. 7. Tom, B. H., and Six, H. R. (eds.), “Liposomes and Immunobiology,” p. 332. Elsevier/North Holland,

New York, 1980. 8. Hale, A. H., Ruebuch, M. K., Harris, D. T., J. Immunol. 125(l), 428, 1980. 9. Herrmann, S. H., and Mescher, M. F., Proc. Nat. Acad. Sci. USA 78(4), 2488, 1981.

10. Hale, A. H., and MC Gee, M. P., Cell Immunol. 58,277, 1981. Il. Humphties, G. M. K., J. Immunol. 126(2), 688, 198 1. 12. Lindahl, K. F., and Bach, F. H., J. Exp. Med. 144, 305, 1976. 13. Peck, A. B., Alter, B. J., and LindahI, K. F., Transplant. Rev. 29, 187, 1976. 14. Camaud, C., Ahman, J., Errasti, P., and Vandeer Gang, R., &and. J. Immunol. 11, 503, 1980. 15. Englehard, V. H., Strominger, J. L., Mescher, M., and Burakoff, S., Proc. Nat. Acad. Sci. USA 75( 1 l),

5688, 1978.

16. Burakoff, S. J., Englehard, V. H., Kaufman, J., and Strominger, J. L., Nature (London) 283, 495, 1980.

17. Math&n, D. J., and Rosenberg, S. A., J. Immunol. 124(5), 2295, 1980. 18. Hayashi, H., Miier, A. L., and Tom, B. H., Afk J. Clin. Expl. Immunol., in press. 19. Miller, A. L., Hayashi, H., andTom, B. H., Fed. Proc. 40, 1041, 1981. 20. Tom, B. H., Rutzky, L. P., Kaye, C. I., Gyasu, R., and Kahan, B. D., In Vitro 12(3), 180, 1976. 21. Tom, B. H., Rutzky, L. P., Oyasu, R., Tomita, J. T., Goldenberg, D. M., and Kahan, B. D., J. Nat.

Cancer Inst. 58(5), 1507, 1977.

22. Ledbetter, J. A., and Hem&erg, L. A., Immunol. Rev. 47,63, 1979. 23. Brunette, D. M., and TiIl, J. R., J. Membrane Biol. 5, 215, 1971. 24. Bangham, A. D., Standish, M. M., and Watkins, J. C., J. Mol. Biol. 13, 238, 1965.

240 RAPHAEL AND TOM

25. Kruski, A. W., and Narayan, K. A., Anal. B&hem. 41,299, 1972.

26. Bartlett, G. R., J. Biol. Chem. 234(3), 466, 1959. 27. Mishell, R., and Dutton, R. W., J. Exp. Fed. 126,423, 1968. 28. Dunn, 0. J., and Clark, V. A., “Applied Statistics: Analyses of Variance and Regression,” pp. 80-

81, 131-142, 337-339. Wiley, New York, 1974. 29. Takasugi, M., and Mickey, M. R., J. Nat. Cancer Inst. 57(2), 255, 1976. 30. Enger, H. D., Thomas, K., Cerottini, J. C., and Brunner, K. T., J. Immunol. 115(2), 356, 1975.

31. Wagner, H., Hess, M., Feldmann, M., and Rollinghoff, M., Transplantation 21(4), 282, 1976. 32. Berke, G., and Schick, B., In (I. P. Witz and M. G. Hanna, Jr., Eds.), “Contemporary Topics in

Immunobiology” Vol. 10, pp. 297-315. Plenum, New York, 1980. 33. Hayry, P., and Anderson, L. C., &and. J. Immunol. 5, 391, 1976. 34. Cerrottini, J. C., and Brunner, K. T., Advan. Immunol. l&67, 1974. 35. Fast, L. D., and Fan, D. P., J. Zmmunol. 120(4), 1092, 1978.

36. Bun&off, S. J., Ratnofsky, S. E., and Benacetraf B., Proc. Nat. Acad. Sci. USA 74(10), 4572, 1977. 37. Kahan, B. D., Rutzky, L. P., LeGrue, S. J., and Tom, B. H., Methods Cancer Res. 18, 197, 1979. 38. Pant, K. D., Dahlman, H. L., and Goldenberg, D. M., Cancer 42, 1626, 1978. 39. Herlyn, M., Steplewski, Z., Herlyn, D., and Koprowski, H., Proc. Nat. Acad. Sci. USA 76(3), 1438,

1979. 40. Magnani, J. L., Brockbans, M., Smith, D. F., Ginsburg, V., Blasczyk, M., Mitchell, K. F., Steplewski,

Z., and Koprowski, H., Science 212, 55, 1981.

Documents

In vitro induction of primary and secondary xenoimmune responses by liposomes containing human colon tumor cell antigens