In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells

CELLULAR

IMMUNOLOGY

28, 371-378 (1977)

In Vitro

Sensitization of Human Lymphoid Cells to Antigens on Cultured Melanoma Cells

I. Culture

Conditions

Resulting

in Augmented

Cell-Mediated

Cytotoxicity

SIDNEY H. GOLUB, PH.D. Division of Oncology, Department of Surgery, UCLA School of Medicine, University of California, Los Angeles, California 90024 Received

Augmt

25,1975

Culture conditions have been established that result in the sensitization of normal human peripheral blood lymphoid cells on allogeneic melanoma monolayers. Optimal culture conditions require 2 to 8 X 10’ mitomycin C treated stimulator melanoma cells to sensitize 5 to 10 X 10’ responder lymphoid cells. Neither rocking nor refeeding of the culture is necessary for the sensitization procedure. Stimulator cells grown in either fetal calf serum or human serum will serve as effective stimulator or target cells. Peak cytotoxic activity was detected at 44 hr in a microcytotoxicity assay, although some cytotoxic activity was detectable at 24 hr.

INTRODUCTION The purpose of this study was to find suitable culture conditions for the in vitro sensitization of human lymphoid cells against antigens found on cultured melanoma cells. It was our intention to establish an allogeneic model system to determine optimal culture conditions for -sensitization prior to investigating autochthonous tumor cell-lymphocyte interactions. Established animal model systems have shown that mixed lymphocyte cultures (MLC) can generate cytotoxic effector cells specific for the histocompatibility antigens of these stimulator cells (1, 2). More relevant to tumor systems described in this report, murine lymphoid cells have been sensitized on xenogeneic or allogeneic monolayers of cultured cells (3, 4). In human systems, not only do standard MLC reactions result in cytotoxic effector cells, but stimulation with allogeneic lymphoid (6, 7) or nonlymphoid tumor cells (8, 9) also results in the development of cytotoxic effector lymphoid cells. This report describes a tissue culture methodology that utilizes human adherent non-lymphoid tumor cells as the antigen to generate sensitized lymphoid cells. MATERIALS

AND

METHODS

Lymphoid cells. Peripheral blood lymphoid cells (PBL) were prepared from heparinized blood samples from healthy volunteers. In order to obtain reproducible stocks of responder PBL, large numbers of PBL were obtained from a single run of an American Instrument Co. “celltrifuge” (Silver Spring, MD). Lymphoid cells

371 Copyright 0 1977by AcademicPress,Inc. All rights of reproduciionin any form reserved.

ISSN 0008~8749

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obtained from the celltrifuge then were further purified on Ficoll-Hypaque gradients and cryo-preserved in liquid nitrogen prior to use. The cryo-preservation method used retained full PBL activity in assays of cytotoxicity and blastogenesis (lo). PBL purified in this manner contained 5 to 15% monocytes and were contaminated with less than 2% granulocytes. Five normal donors were studied and each is identified in the text by a single initial. Ca&Wed &zor ce2l.s. Cultures of human malignant melanoma cells were established by Dr. Hector L. Sulit of the UCLA Division of Surgical Oncolo,qy. The cell lines were maintained in RPMI-1640 medium (Microbiological Associates, Bethesda, MD), supplemented with antibiotics and 20% fetal calf serum or when indicated, with 5% pooled normal AB+ serum (Microbiological Associates) or 2.5 to 5% agamma human serum (Biocelle, Los Angeles, CA). All cultures were used between the 5th and 25th passage. The cell lines were designated as follows: University of California, Los Angeles, Surgical Oncology-M4 (UCLA SO-M4), UCLA SO-MlO, and UCLA SO-M14. For brevity these lines will be referred to in text as M4, MlO, and M14. Sensitization procedure. Details of the requirements for sensitization are presented in the RESULTS section. In summary, tumor cell monolayers were grown in 25 cm2 Falcon tissue culture flasks (Falcon Plastics, Oxnard, CA) in media containing 20% fetal bovine serum. After 24 hr of incubation the cells were treated with 3 to 5 pg/ml of mitomycin C (Nutritional Biochemicals, Cleveland, OH) for an additional 18 hr. The dose of drug required to abrogate DNA synthesis for each cell line was determined. The mitomycin C then was removed by washing the monolayer 5 times with media containing no serum. The responder PBL were added in 5 ml of media containing 20% pooled normal human AB serum and cultures were cultivated for 5 days, except in those experiments on the time requirements for sensitization. PBL were collected from the monolayers by scraping with a rubber policeman, washed once in media containing AB serum, and aliquoted for the blastogenesis and cytotoxicity assays. Control cultures of responder cells cultivated in the absence of sensitizing cells were included in each experiment. Forty to 60% of the original lymphocyte number could be recovered when the optimal sensitization procedure was employed. Assay for cytotoxicity. A modification of the microcytotoxicity test of Cohen et al. (11) was used. In this test, target cells were plated into the wells of a Falcon #3040 Microtest II plate (Falcon Plastics) at a concentration of 3 X lo3 cells/ well. The target cells were allowed to adhere overnight and then were pulse-1abeIed for 18 hr with 0.5 &i/well of [1251]Iododeoxyuridine (IDU) (Amersham/Searle, Arlington Heights, IL) in the presence of 1OM8A4 fluorodeoxyuridine (Roche Laboratories, Nutley, NJ). The target cells then were washed 3 times with media containing 10% fetal bovine serum to remove excess label. Effector cells at concentrations of 1.5 X 105/well in most assays were added in a volume of 0.1 ml/well in media with 20% AB serum. After 24 to 48 hr of incubation at 37”C, the dead target cells and the lymphocytes were removed by washing the plate 3 times. The plate was dried and sprayed with Krylon Plastic Spray (Borden, Inc., Columbu?, OH) and the individual wells were separated using a band-saw. The wells were counted on Nuclear Chicago scintillation counter. Assay for lymphocyte proliferation. Lymphocytes recovered from the sensitizing cultures also were tested for DNA synthesis by a modification of the method of

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Thurman et al. (12). Quadruplicate aliquots of 0.1 ml containing 1.5 X lo6 recovered viable PBL were placed in the wells of a Microtest II plate and 0.5 pCi of [sH]thymidine (New England Nuclear Co., Boston, MA) was added to each well. After 18 hr of incubation, the cells were harvested in a MASH-I automatic multiple harvester (Microbiological Associates) onto strips of fiberglass filter paper, dried, and the individual samples were placed in liquid scintillation fluid for counting in a Beckman liquid scintillation counter (Beckman, Anaheim, CA). Calculation of results. All results are expressed as increments of increased activity when compared to control PBL cultivated for an equivalent period in the absence of any sensitizing cell. Thus, cytotoxicity is expressed as cytotoxic index (CI) calculated by the formula : cI

= (CPM in wells with control PBL) - (CPM in wells with sensitized (CPM in wells with control PBL)

PBL)-

Effector cells were considered cytotoxic if they resulted in a CI of 0.15 or greater that was significant at the 0.05 level by the Student’s t-test. Lymphocyte proliferation, as determined by t3H]thymidine incorporation, is expressed as a response index (RI) calculated by the formula : RI = CPM incorporated by sensitized lymphocytes CPM incorporation by control lymphocytes ’ The data expressed in this fashion, are focused on the increased PBL cytotoxic and proliferative activity due to the sensitization procedure. TABLE

Experiment

1

Dose of

Allogeneic Sensitizing Tumor Cells

Culturea

CPM C3HJTb,”

K.I.

CPM retained in target cells

C.I.

1759 f 115 1835 f 75 1467 i 50 1911f74

-0.00 0.17 -

1

Db D + 0.2 X 10s M4m D+2X106M4m Media alone

1010 f 47 637 f 31 1420 Z+Z 86 64f3

1.00 0.63 1.41 -

2a

D D + 2 X lo8 M14m D + 4 X 10” M14m D+8X 108M14m Media alone

1889 2976 3847 3053 81

f f f f f

86 440 199 154 14

1.00 1.57 2.04 1.62 -

2b

D D f 2 X lo6 M12m D + 4 X lo6 M12m D + 8 X lo6 M12m Media alone

1889 3155 4186 2230 81

f f f f f

86 181 149 93 14

1.00 1.67 2.21 1.18 -

620 f 426 f 400 f 44.5 f 684 f 891 723 588 565 1010

35 27 8 21 44

0.31 0.35 0.28 -

* 31 f 104 f 83 i 185 f 57

0.19 0.34 0.37 -

a 5 X 10’ responder PBL from a single normal donor (“D”) were used in all sensitization cultures. b Cytotoxicity test of effector cells not subjected to in vitro culture (time-zero control) : 2891 i 174 CPM for M4 targets + D; 2861 f 831 CPM for M4 targets + media alone. c CPM of CSH]thymidine incorporated/l X 106 PBL.

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CONCENTRATION OF EFFECTOF? CELLS ( x i05) FIG. 1. Time and dose titration of effector cells from normal donor “D” sensitized on M4 and tested against M4 target cells. Cytotoxic index derived by comparison to nonsensitized PBL. Cytotoxicity assays terminated at 6 (O-----O), 24 (O----O), and 44 (0-O) hours. Effector cell added in serial dilutions starting at 1 X 105/well.

RESULTS Optimal numbers of sensitizing mehnoma cells. Preliminary experiments indicated that 5 to lo6 responder PBL were the maximum number of cells that could be effectively employed in the 5 ml culture system. The optimal number of sensitizing cells was then determined by using replicate cultures of 5 X lo6 responder PBL and various concentrations of stimulator melanoma cells. These results are summarized in Table 1. It can be seen that very low numbers of melanoma cells (2 x 105) were ineffective as stimulators of either lymphocyte proliferation or cytotoxicity. However, if the monolayer contained 2 to 8 X lo6 cells, both proliferative and cytotoxic activity was generated. With the two cell lines employed in this experiment, 4 X log cells was optimal for sensitization, and the system was standardized for this amount. Optimal cytotoxic acivity in the 1251DU assay. Dose and time requirements for the cytotoxicity assay were examined using normal lymphoid cells sensitized on allogeneic melanoma cells. Normal responder PBL (5 x 106) were sensitized on 2 x lo6 M4 cells and tested for cytotoxic activity after 5 days. Effector cells were tested at various concentrations. Replicate target cells were assayed at 6, 24, and 44 hr. The results are shown in Fig. 1. Cytotoxicity is expressed as augmented reactivity when compared to control culture PBL. It was apparent that 6 hr was insufficent for demonstration of the cytotoxic reaction. After 24 hr cytotoxicity was noted but greater activity occurred at 44 hr of incubation with effector cell concentrations of 0.5 X 105/well or more. However, we found that the time varied somewhat with different target cell lines. Most target cells required the 44 hr incubation although a few of the more sensitive ones required only 24 hr to demonstrate differential cytotoxicity between control and sensitized lymphocytes. Based on these

LYMPHOID

CELLS

ON

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TABLE Etfect of Rocking (All Cytotoxicity Experiment No. 1

2

CELLS:

CULTURE

375

CONDJTJONS

2

or Refeeding of Sensitizing Cultures Assays Performed at 1 X 106/Wefl) Average

CPMc

C.I.

Group

Culture

y0 PBL recovered

A

S@ S+M14

40 48

792 f 596 f

82 43

0.25b

B

S S+M14

26 59

787 f 630 f

101 22

0.20

65 66 64

0.14 -

C

S S+M14 Media only

20 21 -

635 f 549 f 821 f

A

S S + Ml4

60 86

728 z?z 58 606 f 28

0.17h

D

S S+M14 Media only

54 65 -

701 * 578 f 752 zt

O.lgh -

a S = PBL from a normal donor (“S”). Cytotoxicity against Ml4 culture of the effector cells (time-zero control) : 1427 f 49 CPM for for Ml4 alone. b P = < 0.05 by Student’s t-test. c Average CPM r2~IDU f SE retained by Ml4 target cells. Group flasks, no refeeding. Group B = cultures in flasks, refed daily. Group refed daily and rocked. Group D = cultures in flasks, rocked.

41 26 95

target cells without in vitro Ml4 + S; 1738 zk 52 CPM

A = stationary cultures in C = cultures in petri dishes,

results, effector cell concentrations of 1 to 2 x lo” were used in all subsequent experiments. This provided an effector: target ratio of approximately 100: 1 for target cells with a plating efficiency of about 50%. Efect of rocking and refeeding the sensitizing cultures. Experiments were performed to determine if sensitization could be augmented by culture conditions for antibody production in vitro as described by Mishell and Dutton (13). Results of experiments of this type are summarized in Table 2. In Experiment 1, 8 x 10s PBL from normal donor “S” were sensitized in the standard fashion in 5 ml cultures in T-25 flasks (groups A). Additional sets of control and experimental cultures were sensitized in T-25 flasks but were supplemented daily with 0.5 ml of RPM1 with 20% human AB serum (Group B). The final pair of cultures were sensitized in 35 mm petri dishes (Falcon Plastics) with a daily supplement of 0.5 ml of fresh media (Group C). This last group of cultures was maintained in a moist chamber on a rocking platform during the sensitization period. Results indicated that the original method employed (Group A) provided the most efficient effector cells in the cytotoxicity assay. Furthermore, the modifications resulted in lower yields of effector cells. In Experiment 2, lymphoid cells were sensitized in the standard fashion (Group A) and replicate cultures were sensitized in the same manner except that they were cultured on the rocking platform (Group D). Again, no marked benefit was obtained by the rocking procedure. Thus, it appeared that neither rocking nor refeeding of the cultures was essential for sensitization or recovery of adequate numbers of required effector cells.

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GOLUB

TABLE

3

Effect of Serum Source Used to Grow Sensitizing C&urea

Effector cells/ well

Target Ml4 (AB) CPM retained

S S + M14(AB) S S + M14(AB) Media alone

2 2 1 1

x 105 x 106 x 105 x 105 -

S S + Ml4(FCS) Media alone

1.5 x 106 1.5 x 105 -

723 281 740 504 760

f f i f f

N.T. N.T. N.T.

113 28 44 67 51

C.I.

0.61” 0.32b -

-

Cells cells Ml4 (FCS) CPM retained 1804 f 1127 f 2087 f 1.501 f 1498 f

C.I.

111 64 221 94 151

0.38” 0.2gb -

792 f 82 596 z!z 43 821 f 64

0.25” -

D PBL from normal donor “S” sensitized on Ml4 cells grown in either AB+ or FCS. C. I. derived by comparison to PBL cultured in absence of Ml4 cells. Cytotoxicity against M14(AB) targets without in vitro culture of effector cells: 692 f 47 CPM for M14(AB) + 2 X lo5 S; 68.5f 91

CPM for M14(AB) + media. b P =
t-test.

eP = < 0.05 by Student’s t-test. N.T. = Not tested. Humn serwn and fetal calf serum grown stimulator cells. Most of the sensitization on allogeneic melanoma cells has been ascribed to histocompatibility antigens although some sensitization may have been due to tumor-associated antigens. However, there was a possibility that the responder PBL were reacting to heterologous antigens introduced from the fetal calf serum (14). Small amounts of fetal calf serum left from the growth media of the stimulator cells could have a mitogenic effect on the PBL that induced nonspecific cytotoxicity (1.5). In order to test for FCS effects on sensitization, stimulator Ml4 cells were grown for 4 weeks in media supplemented with 5% pooled human AB+ serum. Responder PBL from normal donor “S” were sensitized on these AB grown melanoma monolayers, and tested for cytotoxic activity against both AB and fetal calf serum grown target cells. These results are shown in Table 3. For comparison, PBL were also sensitized against FCS grown Ml4 cells in the usual manner. It was clear from these results that AB grown melanoma cells were as effective or perhaps slightly more effective for allosensitization than were the FCS grown cells. Similarly, both FCS and AB grown target cells were sensitive to the cytotoxic effects of PBL sensitized on AB-cultured Ml4 monolayers. DISCUSSION A system has been described for the sensitization of normal human peripheral blood lymphoid cells on allogeneic melanoma monolayers. This system appears to be similar in many respects to the MLC generated cytotoxic effecters cells, and is presumably based on the same types of cell interactions. In this system, we used 5 to IO x lo6 responder PBL for the sensitization procedure. Approximately 60% (from 40 to 75%) of the original number of PBL

LYMPHOID

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could be recovered. These cells were active in cytotoxicity assays against the same type of cell as was used for stimulation. Sensitized cells also showed increased thymidine incorporation indicating a proliferation response in responder cells. Whether this proliferation was necessary for the generation of the cytotoxic effector cells is under investigation. Relatively simple culture conditions were sufficient to generate cytotoxic effector cells on allogeneic tumor cell monolayers. A culture of 6 to 10 X lo6 responder PBL on a monolayer of 4 X lo8 human melanoma cells resulted in increased cytotoxic activity after 5 days of incubation. Neither rocking nor refeeding of the cultures was necessary. However, there was considerable variability in the degree of cytotoxicity induced. In 8 experiments of sensitization against M4 cells, one stock of responder PBL from one donor gave an average cytotoxic index of 0.41 f 11 (standard error). It seems unlikely that this variability was due to a variation in the effector cells employed, because, first, stock and second, the PBL displayed all these cells were from one “celltrifuge” highly reproducible responses to mitogens (10). Thus, the variation was probably related to either the cytotoxicity assay or the sensitization procedure. In order to minimize this problem, only early culture passages of the melanoma cells were used. Because of this variability, greater reliance was placed on a consistent pattern of cytotoxicity or noncytotoxicity than on the magnitude of the cytotoxicity induced. The control, nonsensitized, cultured effector cells demonstrated some cytotoxic activity (Tables l-3). This cytotoxic activity was generally fairly low. For example, with cultured, nonsensitized D effecters (Table 1), cytotoxic indices of 0.08 against M4 targets, 0.09 against Ml4 targets, and 0.12 against Ml2 targets could be calculated by comparison to targets without any added effecters. Effector cells from D showed minimal cytotoxicity against M4 targets (CI = 0.01) when tested prior to sensitization. Maximal cytotoxicity of sensitized effecters against M4 cells could be calculated at CI = 0.23 compared to media controls or CI = 0.17 when compared to the control effecters. Thus, there was a significant augmentation of cytotoxicity by sensitization of D on M4 cells, whether compared against the nonsensitized control D cultures or the reactivity of D effecters prior to sensitization. The reactivity of effecters from normal donor S presented a similar, but somewhat more complicated situation. Against the Ml4 (AB) targets, neither the unsensitized (time-zero) effecters nor the control culture effecters displayed any appreciable cytotoxicity (Table 3). Thus, only the sensitized effecters demonstrated significant cytotoxicity. However, against the Ml4 (FCS) targets, the noncultured effecters displayed a moderate cytotoxic reactivity (CI = 0.18) compared to media controls. This greater cytotoxic effect of PBL from a normal donor on FCS-grown targets was consistent with our previously published finding (16) that one component of “natural” or “nonselective” cytotoxicity was reactivity against fetal calf serum-derived antigens. When PBL were cultured in the absence of any sensitizing melanoma cells, the cytotoxicity obtained was negligible (Tables 2 and 3), and even resulted in enhanced target cell survival in one experiment (Table 3). The exception was Group C (Table 2)) in which the culture conditions employed led to a low recovery, even though cytotoxicity was retained at presensitization levels. Because the background “natural” or “nonselective” cytotoxicity was shown to be affected by in vitro culture conditions (17), we decided to use the control cultured cells to determine the augmentation of cytotoxic activity. However, when the results

378

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were calculated, it was apparent that in vitro co-cultivation of PBL from normal donors with melanoma cells resulted in augmented cytotoxicity. Part I of the study did not address itself to the question of the antigenic specificity of the cytotoxic activity ; that became the subject of Part II. However, the sensitization was not against FCS antigens because the system functioned equally well with human serum-grown cells. The relationship of PBL cytotoxic reactivity from normal donors to the cytotoxicity obtained by in vitro sensitization is unclear. It is possible that the “natural” reactivity of unknown specificity was augmented by or that it preferentially survived the in vitro cultivation with allogeneic tumor cells. This seems unlikely for two reasons. First of all, as shown in the following report, sensitization of PBL from melanoma patients resulted in augmented cytotoxicity that was specific for melanoma targets. Secondly, the “natural” or “nonselective” cytotoxic cell has been shown to be a non-T cell in both mice (18, 19) and man (17, 20), whereas i?z ivtro sensitization is T-cell dependent in almost all systems studied (21). Preliminary results in this system are consistent with a T-cell effector from the in vitro sensitized cultures. ACKNOWLEDGMENTS This work was supported by USPHS Grants CA17013 and CA12582 The author is a recipient of an NIH Research Career Development Award. The author acknowledges the technical assistance of Mr. Douglas Hanson and Mrs. Carol Kelley and the generous provision of “Celltrifuge” samples from Drs. Jack Roth and Yosef Pilch of Sepulveda Veteran’s Administration Hospital, Sepulveda, CA.

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