Int. J . Cancer: 9, 299-304 (1972)

CELL-MEDIATED RESPONSES TO TUMOUR XENOGRAFTS IN MICE

Elizabeth SIMPSON and P. C. L. BEVERLEY Clinical Research Centre, Watford Road, Harrow, Middlesex,

and National Institute for Medical Research, Mill Hill, London, N. W.7, Englatid

A micro-cytotoxic assay has been used to follow the course of the cell-mediated response to a tumour xenograft in normal and ALS-treated mice. In normal mice a peak response was seen at day I0 in both lymph nodes and spleen. Further peaks of cytotoxic activity were seen at days 18 and 42 in the spleen. In ALS-treated mice, in which the tumour grew progressively, the response was slightly delayed in both lymph nodes and spleen. Subsequently 10 out of 12 mice bearing progressively growing tumours, tested at times up to 83 days after tumour grafting, showed immunity in either lymph nodes, spleen or both.

In a previous paper (Beverley and Simpson, 1970) we have described the humoral response to a tumour xenograft in normal and anti- lymphocyte serum (ALS) treated mice. In this paper we describe in vitro studies of lympho- cyte-mediated target-cell killing in similarly treated mice. We have used the micro-cytotoxic test of Takasugi and Klein (1970). The identity of the lymphocyte populations involved was investi- gated. It was hoped that these studies might also throw some light on the more general problem of the growth of highly immunogenic tumours in the face of an immune response.

MATERIAL AND METHODS Mice

adult CBA mice of either sex.

Tumour The hamster tumour line, designated Tr, and

tissue culture methods for its maintenance have been described previously (Beverley and Simp- son, 1970).

For some experiments, large numbers of Tr cells were stored in liquid nitrogen. The freezing mixture used consisted of Eagle's Minimal

All experiments were carried out in young

Essential Medium (MEM) with 15% foetal calf serum (FCS) and 10% dimethyl sulphoxide. One- ml aliquots containing 20 x lo6 cells were slowly cooled with a Linde Biological Freezer.

When required for use the cells were rapidly thawed to 37°C. Cell viability, after recovery from liquid nitrogen, was consistently greater than 90 % by dye exclusion.

Anti-lymphocyte serum ALS was prepared in rabbits by the method of

Levey and Medawar (1 966). The ALS pools used in this study were all shown to prolong the median survival time (MST) of A+CBA skin grafts for more than 22 days (Jooste et al., 1968).

Micro-cytotoxic test The test was performed essentially as described

by Takasugi and Klein (1970). Microtitre plates (No. 3034, Falcon Plastics,

Los Angeles, Calif., USA) were used. The wells were filled with MEM +20 % FCS and incubated overnight at 37" C. (This was found to improve the consistency of plating of the target Tr cells). The medium was removed by inverting and shaking the plates, and each well was refilled with 10 pl of MEM+ZO% FCS containing 100-150 Tr

Received: November 22. 1971.

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cells, by means of a Hamilton dispenser and microsyringe. Plates were placed in a humidified atmosphere of 5 % CO, in air at 37” C and incubated for 4-6 h.

The medium, with any non-attached Tr cells, was then removed by inverting and shaking, and replaced either by 10 pl MEM+20% FCS, or MEM+2O% FCS containing lo5 lymphoid cells (see Fig. 1). The plates were then incubated for 36 h at 37” C in the same humidified gas mixture as before.

After incubation the plates were inverted and shaken to remove medium and lymphoid cells and then flooded with Hank’s Balanced Salt Solution (BSS) and agitated and the medium once more shaken off . The plates were then inspected with an inverted microscope and, if free of lymphoid cells, fixed by flooding with methanol. If lymphoid cells remained they were again flooded with BSS, shaken and inspected. After fixation, plates were stained with Giemsa and the remaining target cells in each well counted using a projection microscope.

Each plate included at least 12 wells with target cells alone. Each plate always included both control and test lymphoid cell suspensions in at least six wells each. Plates were always set up in duplicate.

The percentage cytotoxicity was calculated according to the formula

% cytotoxicity =

(Average No. of Tr cells in wells) (treated with test lymphocytes) x 100 (Average No. of Tr cells in wells)

100-

(treated with control lymphocytes)

The statistical significance of the % cytotox- icity of a test lymphoid cell suspension in comparison with the control was assessed by Student’s t test.

Preparation of lymphoid cell suspensions

Mice were killed by cervical dislocation. External inguinal, brachial and axillary lymph nodes and spleens were removed aseptically and placed in cold BSS containing 100,ug/ml of streptomycin and 100 units/ml of penicillin. Cell suspensions were prepared by teasing in BSS with 21-gauge needles. Cell clumps were allowed to settle and then removed with a sterile Pasteur pipette. The resulting cell suspensions were

100

7 5

> 5 0 u

Y 0 b 0 I- > U 25 9

0

-10

I 5 10 15 20 NUMBER OF L Y M P H O C Y T E S x l o4

FIGURE 1 Optimal lymphocyte/target cell ratio. m-m

Immune spleen cells; 0-0 immune lymph-node cells; 0-0 control spleen cells; 0-0 control lymph-node cells.

Each point represents the mean of the % killing in comparison with target cells alone. All measure- ments were made in six replicate wells.

washed once in BSS and resuspended at room temperature in Haemolytic Gelatin Gey’s Solu- tion. They were then immediately spun down at 120xg, washed once in BSS and twice in MEM+2O% FCS. Finally haemocytometer counts were performed and cell concentration adjusted to lo7 cells/ml.

RESULTS

Optimum lymphocyte target cell ratio In preliminary experiments the number of

lymphocytes required to give optimal specific killing of target cells was studied. Results of a typical experiment are shown in Figure 1. In this experiment varying numbers of pooled 10-day immune or control lymph-node or spleen lymphocytes were assayed for their cytotoxic activity. The results have been expressed as percentage cytotoxicity compared to wells con- taining target cells alone. The Figure shows that 2 x lo5 immune cells give the highest kill but this number of control spleen cells gives an

300

RESPONSE TO TUMOUR XENOGRAFTS IN MICE

TABLE I

PRIMARY CELL-MEDIATED RESPONSE IN LYMPH NODES A N D SPLEEN OF INDIVIDUAL MICE

Day 4 Day 6 Ddy 10 Day 14 Day 18

Lymph - 8.5817.35 32.50k2.90 *** 24.00+ 5.40** 11.171 5.16 8.25k6.58 nodes - 8.17*7.75 39.5012.63 *** 28.83d- 3.01 *** 12.42+ 4.77 12.08&8.96

Spleen 48.25 k3.48 *** 18.75+5.11 * 58.671 6.51 *** 49.42+ 7.68 *** 69.17*2.32 *** 13.12 +9.55 24.55+7.90 ** 55.123 5.43 *** - 8.00+16.24 70.17r4.38 ***

- 16.08t8.87 20.64+5.15 ** 32.441 4.72 *** 3 .501 9.93 -5.17 16 .29

4.2751.66 10.75 $6.35 29.44110.36 * -16.25*10.62 10.75*6.54

I Each figure represents the mean of the ”/. killing in comparison with control cells,

* * = 0.01 < p t0.05; ** = 0.001 < p <0.01; *** = p <O.OOI.

standard error. All measuremenfs made in 12 replicate wells.

unacceptably high background. For this reason we chose to do subsequent experiments using lo5 immune or control cells per well,

Primary immune response

This was studied in three experiments. In Experiment 1, 18 male mice were given 15 x lo6 liquid-nitrogen-stored Tr cells subcutaneously in the ventral midline and a comparable group of control mice were left untreated. At days 4,6 , 10, 14 and 18 thereafter two control and three immunized mice were killed and their spleens and lymph nodes removed for use in the microcyto- toxic test.

In Experiment 2, groups of three female mice were immunized with 13 x 106 of the same batch of liquid-nitrogen-stored Tr cells on days 18, 14, 10 and 6 before assaying their lymphoid organs in the micro-cytotoxic test. All groups and three controls were assayed in one test.

In Experiment 3, groups of three female mice were immunized with l o x lo6 Tr cells as in Experiment 2 but on days 42, 35, 28, 21 and 10 before assaying. Two control mice were used.

The results of Experiment 1 are detailed in Table I. The percentage cytotoxicity of target cells caused by spleen or lymph-node cells from each immune mouse compared to the control mice is shown, together with the standard error.

For the sake of clarity the results of this and Experiments 2 and 3 are shown graphically in Figures 2 and 3. In Figure 2 the development of the cytotoxic response in the lymph nodes is shown. Immunity is first detectable on day 6 and is still strong at day 10, subsequently falling to undetectable levels in most mice by day 18. In the

spleen, Figure 3, a single mouse showed significant immunity at day 4 and three out of six at day 6 . The peak response was observed at day 10. There is a statistically significant fall in the response at day 14 followed by a second peak in some mice at day 18. There is a second fall at days 21 and 28 and another rise between days 35 and 42.

10(

7 5

5 c > k u 2 p 2 5 > U -

0

-2 5

10 2 0 30 4 0 DAYS

FIGURE 2 Primary cell-mediated response, lymph nodes.

m represents the % cytotoxicity of lymphocytes from one mouse. A- A represents the mean cyto- toxicity of lymphocytes from mice tested on the same day of the response.

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SIMPSON AND BEVERLEY

Long-term tumour bearers

As described in a previous paper (Beverley and Simpson, 1970) mice treated for a limited period with ALS and grafted with Tr cells, in a percentage of cases show progressive growth of tumour. In the present experiments groups of male or female mice were given three doses of 0.25 ml of ALS, subcutaneously on alternate days, before grafting with 3-10 x lo6 Tr cells also subcutaneously. The majority of mice developed slowly-growing progressive turnours but survived for as long as 100 days after tumour grafting. All animals tested for cell-mediated immunity bore tumours at the time of killing. Mice were killed at intervals during and after ALS administration and their spleens and lymph nodes used in the micro-cytotoxic test. In each experiment a group of mice was also treated with ALS but not grafted with Tr. Lymphocytes from these animals were used as controls in each micro-cytotoxic test. The results of these experiments are shown in Table 11.

1oc

7 5

5 0

> t u X

2 5 > V *

0

-2 5

-6 0'

I 10 2 0 3 0 4 0 DAYS

FIGURE 3 Primary cell-rnediated response, spleen. rn repre-

sents the % cytotoxicity of lymphocytes from one mouse. A- A represents the mean cytotoxicity of lymphocytes from mice tested on the same day of the response.

This shows that during ALS administration immunity can be detected in the lymph nodes and spleen but the response is delayed in comparison with that in normal mice (Fig. 2, 3). There is a statistically significant promotion of target cells by spleen cells from day 14 ALS- treated tumour-bearers. After cessation of ALS treatment 10 out of 12 mice tested at various times showed immunity in either lymph nodes or spleen. There does not appear to be any correlation between the response found in the two lymphoid organs. The finding of significant immunity in the lymph nodes of five mice bearing tumours, as long as 83 days after Tr inoculation, contrasts with the situation in normal mice, in which no detectable immunity was found after 18 days.

DISCUSSION

We have investigated the in vitro cytotoxic responses of normal and ALS-treated mice. In previous work there is evidence for cytotoxicity of both bone-marrow-derived and thymus- derived lymphocytes. In the rat xenograft system described by Harding et al. (1971) killing has been clearly shown to be mediated by non-immune bone-marrow-derived lymphocytes and to require the presence of antibody to the target cells. In contrast there is direct evidence for the role of thymus-derived lymphocytes in allograft rejec- tion (Cerottini et al., 1970). The evidence for involvement of thymus-derived cells in xenograft rejection is indirect, bascd on the ability of ALS to maintain xenografts of skin (Lance and Medawar, 1968) or tumour (Phillips and Gazet, 1968). The fact that in our system also, ALS allows growth of a xenograft, without greatly affecting the antibody response to it (Beverley and Simpson, 1970) suggests that thymus-derived cells play an important role in the normal rejection process. Direct evidence for this has been obtained with the use of anti-theta allo- antiserum, which abrogates the cytotoxicity of immune lymphocytes (Beverley and Simpson, in press). This is evidence for involvement of thy- mus-derived lymphocytes (Schlesinger and Yron, 1969; Raff, 1969).

The response in normal mice shows some interesting features. The lymph-node response is monophasic, suggesting that immune cells leave

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RESPONSE TO TUMOUR XENOGRAFTS IN MICE

the node after day 10. In the spleen several peaks of cytotoxicity are seen. The first two, seen at days 10 and 18, are similar to those found by Denham et a/. (1970). However, in their system the depression at days 21 and 28 was not seen. Denham et a/. suggest that their two peaks represent the activity of two populations of cells which differ in radiosensitivity and other properties. We have no information on the properties of the cells involved in the different phases of the response except the sensitivity of the early peak (day 10) to treatment with anti- theta (see above).

Cyclical phenomena have been reported in humoral responses (Moller et al., 1968) and it has been suggested that these are due to antibody- mediated feedback mechanisms. Similar controls may perhaps operate in lymphocyte cytotoxic responses, mediated either by antibody (or antibody-antigen complexes) or by some other mechanism. In this system antibody is present until at least day 70 following inoculation of Tr cells (Beverley and Simpson, 1970).

In ALS-treated mice grafted with tumour cells we have consistently detected lymphocyte- mediated cytotoxicity. Even during the course of ALS administration both lymph nodes and spleen showed cytotoxicity, although this was delayed in comparison with normal mice given tumour alone. This cytotoxicity is surprising in view of the known effect of ALS in depleting thymus-dependent areas in lymph nodes and spleen (Lance, 1968; Simpson and Nehlsen, 1971). Since at this time the tumour is showing progressive growth in the face of demonstrable immunity, it seems likely that the ALS prevents sensitized circulating lymphocytes from reaching the tumour.

However this explanation would not account for progressive growth of tumours long after cessation of ALS treatment. At this time also, the majority of mice show high levels of cell- mediated cytotoxicity in either or both lymph nodes and spleen. It may be that the delay in development of a cytotoxic response, seen in the ALS-treated mice, is sufficient to allow establish- ment of the tumour. One explanation for continued tumour growth after cessation of ALS would be that the tumour simply outgrows the host immune response. Another explanation would be the presence of enhancing antibody (Hellstrom and Hellstrom, 1970). We have

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previously failed in this system t o demonstrate ACKNOWLEDGEMENTS

such antibody by in vivo passive transfer experiments. Attempts to demonstrate blocking This work was carried ou t during the tenure by in vitro are in progress. T h e fact t ha t sera f rom P.C.L.B. of a n M R C Junior Research Fellow- long-term tumour bearers are known to contain ship. cytotoxic antibodies (Beverley a n d Simpson, W e would like t o thank Mr. P. R. Chandler 1970) does not rule ou t enhancement. for patient and skilful technical assistance.

REPONSES A M E D I A T I O N C E L L U L A I R E X E N O G R E F F E S T U M O R A L E S CHEZ L A S O U R I S

Une kpreuve microcytotoxique a 6th utiliske pour suivre le cours de la rkponse a mkdiation cellulaire a une xhnogreffe tumorale chez des souris normales ou trait& a I‘ALS. Chez les souris normales, une rkponse maximale a Ptk observke le loe jour dans les ganglions lymphatiques et la rate. D’autres maximums d’activitk cytotoxique ont ktt constatks les 1 8 e et 42e jours dans la rate. Pour les souris traitkes a I’ALS, chez lesquelles la tumeur se diveloppait progressivement, la rkponse a ktk Ikg2rement retardke dans les ganglions lymphatiques et dans la rate. Par la suite, I 0 des 12 souris porteuses de tumeurs se divrloppant progressivernent, qui avaient ktk testkes jusqu’a 83 jours aprks la grefe tumorale, ont fait preuve d’une immunite qui a e t t observee dans les ganglions lymphatiques etlou dans la rate.

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CEROTTINI, J.-C., NORDIN, A. A., and BRUNNER, K. T., In vitro cytotoxic activity of thymus cells sensitized to alloantigens. Nature (Lond.), 227, 72-73 (1970).

DENHAM, S., GRANT, C. K., HALL, J. G., aild ALEXANDER, P., The occurrence of two types of cytotoxic lymphoid cells in mice immunised with allogeneic tumor cells. Transplantation, 9, 366-382 (1970).

HARDING, B., PUDIFIN, D . J., GOTCH, F., and MACLENNAN, I. C. M., Cytotoxic lymphocytes from rats depleted of thymus processed cells. Nature New Biology, 232, 80-81 (1971).

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