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(1990)

Effects of Splenectomy on the Development of Tumor-Specific Immunity RODERICH E. SCHWARZ, M.D., Departments

of *Surgery

PH.D.,* AND JOHN C. HISERODT,

M.D.,

and tPathology, University of Pittsburgh School of Medicine, Pittsburgh, and the Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

PH.D.? Pennsylvania

15260,

Presented at the Annual Meeting of the Association for Academic Surgery, Louisville, Kentucky, November 15-18, 1989

We have previously reported on the antimetastatic effects of experimental adoptive immunotherapy using plastic adherent lymphokine-activated killer cells (ALAK) cells (R. E. Schwarz et al. Cancer Res. 49: 1441, 1989). We have also reported that the spleen is a superior source of lymphocytes for A-LAK cell generation (R. E. Schwarz and J. C. Hiserodt, Med. Hypotheses 28: 165, 1989). This study, therefore, was designed to examine the effects of splenectomy itself on tumor growth in an experimental animal model. Natural killer (NK)-resistant MADB106 mammary adenocarcinoma cells were injected iv into F344 rats to generate multiple lung metastases. Splenectomies (Sx) were performed on Days -6, -3, -1, 0, 1,3,6, and 10, counted from the time of tumor injection. Groups consisted of six animals each, and shamanesthetized and -operated animals served as controls. Splenectomies, if performed between Days -3 and +l, had significant antitumor effects as documented by the number of outgrowing surface metastases (6 + 7 vs >300; P < 0.0001) and by animal survival (>lOO vs 21 + 3 days; P < 0.001). However, splenectomies, performed at an earlier or later stage, did not show these effects. Sx did not alter peripheral blood NK activity or the percentages of mononuclear cell subsets except for a slight decrease in the T-helper/T-suppressor ratio (P < 0.04). Interleukin 2 (rhIL2), given at 2.5 X 10’ U/kg/day for 3 days immediately after splenectomy, completely abrogated the observed antitumor effects. Subcutaneous tumor rechallenge of long-term surviving animals showed no tumor take in 87% of the animals. We conclude that the development of tumor-specific immunity in this model is facilitated, if splenectomy is performed during a critical interval, and is abrogated by IL2 administration. Splenectomy can show significant immunomodulatory and/or therapeutic effects in tumor-bearing individuals. 0 1990

Academic

Press,

Inc.

INTRODUCTION

AND METHODS

Animals Male Fischer 344 rats (75-100 g) were purchased from Taconic Farms (Germantown, NY) and housed in a specific-pathogen-free animal facility at the Pittsburgh Cancer Institute. and Tumor Cells

Standard culture medium (CM) consisted of RPM1 1640 (GIBCO, Grand Island, NY) supplemented with 10% 448

Inc. reserved.

MATERIALS

Culture Medium

Adoptive immunotherapy of cancer (AIT) using lymphokine-activated killer cells (LAK cells) has resulted in 0022-4804/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

promising results in patients with certain types of tumors in advanced stages [l-3]. In uitro culture of peripheral blood mononuclear cells (PBMNC) with interleukin 2 (IL2) over several days can render several lymphocyte subsets capable of mediating direct, non-MHC-restricted antitumor cytotoxicity [4,5], and it has been shown that the predominant in vitro LAK activity is mediated by the IL2-activated natural killer (NK) cell [6]. Our lab has developed a technique for separating NK cells from PBMNC cultures in IL2 by means of plastic adherence [ 71. These adherent LAK cells (termed A-LAK cells) mediate superior in vitro [7] and in uiuo [8] antitumor activity and have successfully been cultivated from PBMNC of normal humans [9] and patients with several types of malignancies [lo, 111. We have also shown that removal of the spleen as the source of mononuclear cells (splenic MNC) for A-LAK cell generation rather than PBMNC results in increased A-LAK cell numbers and per cell lytic activity in both animals [12] and humans (own results, submitted for publication). It seemed logical, therefore, to design experiments that investigate the feasibility of using the tumor-bearing individual’s own spleen for the generation of splenic A-LAK cells for subsequent cellular AIT as proposed earlier [ 121. The results show that splenectomy itself can initiate the development of specific antitumor activity and lead to long-term cure and resistance of animals injected with lethal numbers of tumor cells if performed at a certain, early time point before or shortly after tumor cell exposure.

SCHWARZ

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FCS, antibiotics (streptomycin/penicillin), and L-glutamine. The tumor used for in vivo experiments consisted of the NK-resistant mammary adenocarcinoma MADB106 syngeneic in F344 rats. The murine mastocytoma, P815, was also used as an indicator of LAK activity, and the Moloney virus-induced YAC-1 T cell lymphoma was used as the indicator of NK activity. All tumor cells were grown in standard medium and subcultured three times a week. Preparation

of PBMNC

Blood was taken from animals at different time points after splenectomy or after sham operation. Peripheral blood mononuclear cells were obtained after centrifugation on Ficoll-Hypaque gradients (density = 1.077) at 400g for 20 min. The cells were harvested from the interface, washed 2X in CM and used for flow cytometry studies or cytotoxicity assays. Flow Cytometry A panel of antibodies was used in these studies. These included the mouse IgGl monoclonal antibodies OX19 (CD5), OX8 (CD8), W3/25 (CD4), and OX41 (CD14), all purchased from Accurate Scientific (Westbury, NY). Each of these antibodies was used at 1:lOO dilution based on preliminary dose-response titrations. Anti-asialo-GM1 and LyIgG were purchased from Wako Chemical Co. (Dallas, TX) and used at a 1:200 dilution. All second step reagents were fluorescein isothiocyanate-labeled F(ab’)z fragments of goat antibody against the primary antibody and were purchased from Cappel (Malvern, PA). For surface marker analysis, 2 X lo5 lymphocytes were placed in 12 X 75-mm glass tubes in 0.1 ml of staining buffer (phosphate-buffered saline (pH 7.3):0.1% sodium azide:2% FCS). Various antibodies were added to the cells for 30 min at 4°C. The cells were washed twice, incubated with fluorescein isothiocyanate-labeled second antibodies, washed, and resuspended on 1% paraformaldyhyde prior to analysis for fluorescence on a FACStar flow cytometer (Becton-Dickinson, Mountain View, CA). Cytotoxicity

Assay

Cytotoxicity was measured in a standard 4-hr 51Cr release microcytotoxicity assay using 96-well, round-bottom microplates (Costar, Cambridge, MA). The target cells were labeled with 100 &i of Na251Cr04 per 2 X lo6 cells, washed, and seeded into 96-well culture dishes at 5 X lo3 cells/well. Suspensions of effector cells were then added to triplicate wells to give various effector:target ratios in a final volume of 200 ~1. After an additional incubation at 37°C for 4 hr, 100 ~1 of supernatant was removed from each well and counted in a gamma counter to determine experimental release (ER). Spontaneous release (SR) was obtained from wells receiving target cells and medium only, and total release (TR) was obtained from wells re-

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ceiving 1% Triton X-100. The percentage of cytotoxicity was calculated by: % of cytotoxicity

ER-SR = TR _ SR X 100.

Lytic units of cytotoxic activity were determined from linear regression curves plotted from various effector:target ratios. In all cases, 1 lytic unit was defined as the number of effector cells required to cause 20% specific 51Cr release from 5 X lo3 target cells. Total lytic units per culture were calculated by adjusting the lytic unit&O7 cells value to the total number of cells in the culture. Splenectomy Splenectomies were performed on Days -6, -3, -1, 0, 1, 3, 6, and 10, counted from the time of tumor injection. Groups consisted of six animals each, and sham-anesthetized and -operated animals served as controls. For splenectomies, rats were anesthetized with Fluothane inhalation, and the spleen was exposed through a left subcostal lateral laparotomy. Hilar vessels were clamped, the spleen was removed, and the vessel stump was ligated with 3-O silk suture. Skin staples were used for wound closure. Sham operations consisted of laparotomy, mechanical manipulation of the spleen, and wound closure with skin staples. Tumor Cell Injections Pulmonary metastases. F344 rats received iv injections in the lateral tail vein with 7 X lo5 viable MADB106 tumor cells in 1 ml of RPMI. Similar to the splenectomies, tumor cell injections were done under Fluothane inhalation anesthesia. After 18 days the animals were sacrificed by CO2 asphyxiation, the lungs were inflated with India ink and bleached in Fekete’s solution, and the pulmonary metastases were counted and photographed. For survival studies, all animals were autopsied to determine the extent of metastatic disease. Subcutaneous tumor. For subcutaneous tumor rechallenge in long-term survivors after splenectomy, 1 X lo6 viable MADB106 tumor cells in RPM1 were injected into the dorsolateral skin fold. Animals were examined for subcutaneous tumor growth and, in the case of tumor development, the tumor growth rate was assessed by measuring the tumor size. Animals were sacrificed once the tumor had reached a size of 9 cm3 or larger. Statistics Statistical analysis of the differences between splenectomized groups was performed using the nonparametric Kruskal-Wallis test. Pairwise comparisons between individual groups were done with Mann-Whitney rank sum tests. P < 0.05 was considered significant.

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FIG. 1. Lungs from tumor-bearing and splenectomized animals. The lungs were harvested 18 days after tumor cell injection and are representative of the following groups: splenectomy 3 days before tumor injection (upper left), splenectomy at the time of tumor injection (upper middle), splenectomy 3 days after tumor injection (upper right), and sham-operated control (lower).

RESULTS

Intravenous injection of 7 X lo5 MADB106 tumor cells led to the death of all F344 rats before Day 24 after injection. If splenectomy was performed within 3 days prior to or 1 day after tumor cell injection, animals developed significantly fewer numbers of outgrowing Pulmonary surface metastases (Fig. 1, Table 1). For these groups,

TABLE Effect

Sx Sx Sx Sx Sx Sx Sx Sx

None Day Day Day Day Day Day Day Day

1

of Splenectomy on the Number Pulmonary Metastases

Treatment group

-6 -3 -1 0 1 3 6 10

Mean number metastases + SD >300 285 f 72 5+ 7 10 -t 12 4+ 5 15* 7 >300 >300 >300

animal survival was extended to beyond 100 days, an interval after which long-term cure can be safely assumed (Table 2). Compared to &am-anesthetized and -operated controls, reduction in the number of lung metastases and prolongation of survival were statistically highly significant (Tables 1 and 2). In the four groups of rats, in which splenectomy was performed on Day -6, 3, 6, or 10, no

TABLE of Outgrowing

Significance difference

WA N.S.

P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 N.S. N.S. N.S.

Effect

of

Treatment group None SE Day Sx Day Sx Day Sx Day Sx Day Sx Day Sx Day Sx Day

-6 -3 -1 0 1 3 6 10

2

of Splenectomy on Survival Tumor Cell Injection

Mean survival in days + SD 21f 2 23+ 5 89 * 21 1100 93 + 24 78 + 19 20f 4 22f 3 18+ 5

Long-term survivors (>lOO days)

‘W O/f3 5/6

6/6 5/e 5/6

O/6 O/6 Of6

after

Significance of difference

N/A

N.S.

P < 0.001 P < 0.001 P < 0.001 P < 0.001 N.S. N.S. N.S.

SCHWARZ

TABLE

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TABLE

3 Effect

Effect of Splenectomy on the Composition of Peripheral Blood Mononuclear Cell Subsets % of positive cells (median of four rats per group)

of Interleukin Induced

Treatment

Antibody used for flow cytometry

Normal control

1 day post-Sx

3 days post-Sx

6 days post-Sx

0x19 OX8 W3/25 0x41 Asialo GM, lyIgG CD4/CD8 ratio

53 23 37 8 21 10 1.6

57 27 30 5 17 11 1.1

56 31 31 7 23 8 1.0

54 29 34 5 18 9 1.2

such effect on tumor reduction or survival extension could be noted. For all these groups, changes in metastases number and survival time were not significantly different from those of the sham-operated controls (Tables 1 and 2). In order to examine the mechanisms of this splenectomy-induced antitumor effect, we studied possible changes in numbers or function of mononuclear cells that were capable of mediating cellular antitumor responses. Flow cytometric analysis of peripheral blood mononuclear cell subset frequencies is listed in Table 3. As can be seen, there were no significant changes in PBMNC subsets between groups of controls and splenectomized animals when tested at different time points after splenectomy. There was also no obvious difference in cytotoxic activity after splenectomy between rats that carried tumors and those that were tumor free. The only difference between Day -1 splenectomy and controls was a decreased Thelper/T-suppressor (Tn/Ts) ratio (P < 0.04; Table 3). Spontaneous non-MHC-restricted cytotoxicity (NK activity) of freshly isolated PBMNC was not changed after splenectomy in the presence or absence of tumor (Table 4). Cytotoxic activity against NK-resistant target cells (lymphokine-activated killer activity) could not be detected in PBMNC after splenectomy (Table 4). In the absence of obvious changes in the cellular composition of PBMNC after splenectomy, experiments were

Control IL2 Days IL2 Days Sx Day 0 Sx Day 0 Sx Day 0

group

5

2 Treatment on SplenectomyTumor Reduction Mean survival in days + SD 21rf: 24+ 19* 93 + 22f 48 t-

O-2 8-10 + IL2 Day 0 + IL2 Day 8

2 5 3 24 4 31

Significance of difference N/A N.S. N.S. P < 0.001 N.S. P < 0.03

done to study the possible role of cytokines in mediating a postsplenectomy antitumor effect. For this purpose, animals were treated with 2.5 X lo5 U/kg/day human rIL2 for 3 days ip following splenectomy. Although IL2 treatment alone did not affect tumor growth or long-term survival compared to control rats, early IL2 administration after splenectomy (Day 0) completely reversed the splenectomy-related antitumor effect (Table 5), while late (Day 8) IL2 treatment did affect the splenectomy effect to a minor extent as measured in survival after tumor cell injection. All animals that showed long-term survival after tumor cell injection and splenectomy were challenged with a subcutaneous injection of 1 X lo6 MADB106 tumor cells. With only one exception, all animals tested showed no evidence of tumor growth within 100 days postchallenge, while age-matched controls developed palpable SCtumor nodules within 4 weeks (Table 6). DISCUSSION

The spleen as the major cell reservoir of the immune system has been used routinely in murine experiments as the source of cells for in vitro activation and subsequential cellular (adoptive) immunotherapy of cancer [13-E]. Purified IL2-activated NK cells have been shown to be

TABLE

6

Tumor Growth after Subcutaneous Rechallenge Splenectomized Long-Term Tumor Survivors TABLE Spontaneous

Cytotoxic

in

4 Activity

of PBMNC Group

Number of long-term survivors

Tumor growth after subcutaneous rechallenge

Lytic units 20/107 cells against target Group

YAC-1

P815

Normal control 1 day post-Ss 3 days post-Sx 6 days post-Ss

110 89 123 95

24 28 25 31

MADB106 4 2 6 3

Untreated Control Sx Day -3 Sx Day 0 Ss Day 3 Sx Day 0 + 112 Day 0 Sx Day 0 + IL2 Day 8

N/A 0 5/6 516 0 0

2/6

6/6 WA

O/6 O/5 N/A N/A

l/2

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more effective in mediating antitumor cytotoxicity in vitro [7] and in uiuo [8] compared to conventional lymphokineactivated killer cells which, for treatment in humans, are usually derived from unseparated PBMNC cultures. Since our results demonstrated the spleen as a superior source for ILB-activated NK cells (A-LAK cells), it appeared logical to investigate the feasibility of splenectomy in tumor-bearing individuals as a source of cells for adoptive immunotherapy. The data presented show a strong antitumor effect of splenectomy itself leading to complete cures of many animals. This effect, however, was confined to a narrow time span before and shortly after initial tumor cell injection. Although several immunological consequences of splenectomy are known, including increased susceptibility to infections, especially pneumococcal infections [16], a decrease in serum immunoglobulin and transferrin levels [17], and an increased release of lymphocytes from the thymus [ 181, the impact of splenectomy on the progression of a cancerous disease is not well documented. Although splenectomy has been shown to potentiate the induction of malignant, but not benign, colorectal tumors in rats [ 191, the same procedure seems to not change or even decrease the risk of healthy individuals developing cancer in later life [20, 211. In tumor-bearing animals, removal of the spleen has indeed demonstrated obvious beneficial effects such as the limitation of tumor cell growth in secondary sites [22,23], improved survival [24], and enhanced peripheral blood natural killer cell activity [al]. Yet, those improvements seem to depend on the time interval between surgical excision of the primary tumor and the splenectomy and on the total tumor mass at the time of operation [25]. B-cell leukemia growth is, for instance, inhibited and overall survival improved if a splenectomy is performed 1-3 days after tumor inoculation, while splenectomy 8 days after tumor inoculation in the same model does not improve long-term survival [26]. Splenectomy in other experiments had no effect on the frequency of pulmonary metastases of B16-FlO murine melanoma with a time interval of 2 weeks between splenectomy and tumor cell injection [27] and did not influence peritoneal tumor growth when performed 4 days earlier [28]. For humans with cancer, there may be similar benefits and an improved prognosis by extirpation of the spleen, if done at a relatively early stage [29,30], although it should be noted that there are reports favoring spleenpreserving operations in gastric cancer patients without splenic hilar lymph node metastases [31]. Most of these references seem to confirm the reported evidence that splenectomy can mediate beneficial antitumor effects primarily if performed at an early stage of tumor development, but will not influence or may even promote tumor growth if performed at a time of advanced tumor establishment. A similar heterogeneous and time-dependent effect of splenectomy seems to exist in organ transplantation, where this procedure may either enhance graft tolerance [32, 331 or, especially in combination with

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chemical immunosuppression such as cyclosporine A, lead to reduced graft survival [33]. The reason for this appears to be the possibility that different components of the Tcell system, which is predominantly involved in antitumor activities [34], are unequally affected by splenectomy, e.g., there is a stronger impairment of T-helper/T-amplifier cells than of T-suppressor cells especially in the first week after splenectomy [35]. This suggestion offers a possible explanation for the findings in this study. The fact that the development of specific immunity after splenectomy can be inhibited by giving IL2, i.e., a T-helper function, makes the impairment of the T-helper system with insufficient cytokine production and resulting lack of suppression after removal of the spleen a likely candidate to cause the observed antitumor effect. The slight change in T-helper/T-suppressor ratio as observed here may represent a compensatory attempt by the organism. Since splenectomy at any time did not cause major changes in NK cell activity or in the composition of PBMNC subsets, these factors do not seem to be crucially involved in the splenectomy-induced antitumor effect. One possible group of cells that might be of importance, however, is that of monocytes/macrophages. It has been noted before that after splenectomy, Kupffer cells alter their response to endotoxin [36]. While this response is hyperactivated during the first 2 postsplenectomy days, it is depressed during the following weeks. We therefore are tempted to speculate that in the reported system, postsplenectomy activation of macrophage antigen presentation and removal of T-helper cells with subsequent decreased T-suppressor cell function facilitate the development of tumor-specific immunity. More detailed humoral and cellular mechanisms and the validity of these findings for other experimental tumor models are currently under investigation in our laboratory. REFERENCES 1.

Rosenberg, S. A. Development of new immunotherapy for the treatment of cancer using IL2. Ann. Surg. 208(2): 121,1988.

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West, W. H., Taner, K. W., Yannelli, J. R., Marshall, G. D., Orr, D. W., Thurman, G. B., and Oldham, R. K. Constant-infusion recombinant interleukin 2 in adoptive immunotherapy of advanced cancer. N. Engl. J. Med. 316: 898,1987.

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