The Japanese Journal of Surgery (1992) 22:35-39

~ S U R G E R Y T O D A Y

© Springer-Vedag 1992

Two-Color Flow Cytometric Analysis of Splenic Lymphocyte Subpopulations in Patients with Gastric Cancer

MAKOTO TAKAHASHI, 1 SHIGERU FUJIMOTO, 2 MITSURU TAKAI, KAZUHIDE OHNO, FUMIO ENDOH, YOSHIO MASUDA, YOSHINORI MASUDA, and GOROH OBATA 1

1 The Division of Surgery, Matsudo Municipal Hospital, Matsudo, Japan and z The First Department of Surgery, School of Medicine, Chiba University, Chiba, Japan

Abstract: Lymphocyte subpopulations of the spleen were assayed in 26 patients with gastric cancer and 5 patients with benign disease using two-color flow cytometric analysis. The ratio of Leu 2a+ • Leu 15+ cells, or suppressor T cells, in the gastric cancer patients was about 6 per cent, being higher than that in the patients with benign disease (p < 0.05). There were fewer Leu 7+ -Leu 11- cells, or natural killer -NK-cells, in the gastric cancer patients in stage III or IV than in those with stages I or II (p < 0.05). The ratio of Leu 3a+ • Leu 8 - cells, or helper T cells, in the stage IV patients accounted for about 15 per cent of the splenic lymphocytes, which was less than that seen in the patients in stages I or II (p < 0.05). The ratio of Leu 2a+.Leu 15- cells, or cytotoxic T cells, was approximately twice that of suppressor T cells. The pre-operative administration of lentinan plus OK-432 increased the ratio of Leu 4+ • HLA-DR+ cells, or activated T cells, and cytotoxic T cells (p < 0.05 and p < 0.01, respectively). The above results suggest that lymphocyte subpopulations in the spleen may have more immuno- suppressive potential in proportion with the stage of gastric cancer, but that this reduced immune state may be altered when lentinan and OK-432 are given to these patients.

Key Words: splenic lymphocyte subpopulation, gastric cancer, two-color flow cytometry

Introduction

Splenectomy forms part of the surgical treatment for patients with advanced gastric cancer, the objective being to eradicate metastatic lymph nodes in the splenic hilar region. However , there is a controversy concerning removal of the spleen as this organ contains large numbers of lymphocytes. Toge et al. 1 reported

Reprint request to: Makoto Takahashi MD, The Division of Surgery, Matsudo Municipal Hospital, 4005 Kamihongou, Matsudo, Japan (Received for publication on Sep. 14, 1990)

that maturation of suppressor cells occurs in the spleen, after which they migrate to the peripheral blood. Miwa et al. 2 described the clinical efficacy of splenectomy for advanced gastric cancer as deduced from data on survival rates.

Advances in immunologic studies on monoclonal antibodies against T cells have led to effective methods of examining cell function and making quantifying estimations. Two-color flow cytometry has made it possible to perform a reliable and effective analysis of a cell subpopulations. 3-8 In the current study, the lymphocyte subpopulations and effects of the pre- operative administration of a biological response modifier (BRM) on the ratios of splenic lymphocyte subpopulations using two color flow cytometry are reported.

Materials and Methods

Spleens were obtained from 26 patients with gastric cancer, including 17 males and 9 females with a mean age of 54.6 _+ 11.7 years, and 5 patients With benign disease, including 5 females with a mean age of 46.0 _+ 18.9 years.

The clinical stage of the gastric cancer patients, given in Table 1, was in accordance with the General Rules for the Gastric Cancer Study in Surgery adopted by the Japanese Society for gastric cancer. 9 For a control, data on 2 patients with portal hypertension, one with idiopathic thrombocytopenic purpura and 2 with a splenic cyst were used.

Cell Preparation

Approximately 5 g taken from the excised spleen was teased through stainless mesh, filtrated into single cell suspension in RPMI 1640 medium, after which mono- nuclear cells were isolated by Ficoll Hypaque gradient centrifugation and suspended at 1 × 106 cells/ml.

36 M. Takahashi et al.: Splenic Lymphocyte Subpopulations

Table 1. Clinical characteristics of gastric cancer patients with or without the pre-operative administration of a biological response modifier

BRM group Control group

Clinical stage I,II III IV I,II III IV

No. of patients 3 3 7 Median age (yrs) 59 51 51 Sex (male/female) 2/1 3/0 3/4 BRM

Lentinan alone 1 1 2 OK-432 alone 1 1 1 Lentinan plus OK-432 1 1 4

4 5 4 56 61 52

2/2 4/1 3/1

Group 1: Group 2:

Group 3:

Group 4:

13 patients not given BRM. 4 patients given 4 mg of lentinan intravenously twice per week pre-operatively. 3 patients given subcutaneous injections of OK-432 1, 3, and 5 KE, every other day pre-operatively. 6 patients given a combination of lentinan and OK-432.

Details of the clinical stages in each group are given in Table 1. The statistical difference was analysed by Student's t-test.

Reagents

The monoclonal antibodies against T lymphocytes included the Leu series (Becton-Dickinson Corp., Mountain View, CA, USA). The combination of anti- bodies for two-color double staining was as follows: Leu 3a × Leu 8, Leu 4 × Leu HLA-DR, Leu 7 × Leu 11, and Leu 2a × Leu 15.

Techniques

Twenty-five gl of medium containing 1 x 106 cells/ml were incubated at 4°C for 30rain with 5 gl aliquots of monoclonal antibodies labelled by fluorescein isothiocyanate and phycoerythrin fluorescein. Fluores- cein-conjugated, or two-color stained subpopulations of cells were determinded by a FACS (Fluorescent Activated Cell Sorter) 440 analyser (Becton-Dickinson Corp., USA).

Pre-operat ive Admin is t ra t ion o f B R M

The 26 patients with gastric cancer were divided into the following 4 groups:

Results

Splenic L y m p h o c y t e Subpopula t ions o f Patients with

Gastric Cancer

The splenic lymphocyte subpopulations in each stage of gastric cancer are shown in Table 2. The ratio of Leu 3a+ • Leu 8+ cells, or suppression inducer T cells, 3 was higher in the stage IV patients than in the stage I l l patients (p < 0.10). The ratio of Leu 3 a + . Leu 8 - cells, or helper T cells, 4 was lower in the stage IV patients than in those in stages I or II (p < 0.05). Leu 7+ • Leu 1 1 - , Leu 7+ • 11+ and Leu 7 - • 11+ cells are functionally NK cells, 5 and the ratio of Leu 7 + . Leu 1 1 - cells was higher in the patients in stages I and II than in those in stages II or IV (p < 0.05 and p < 0.10, respectively).

Other subpopulations showed no significant differ- ence in ratios among the 4 stages. Leu 4+ • H L A - D R - cells, or pan T cells, comprised about 30 per cent and Leu 4 - • H L A - D R + cells, or pan B cells, 50 per cent of the splenic lymphocytes. The ratio of leu 2 a + . Leu 15+ cells, or suppressor T cells, 6 was approximately half that of the Leu 2a+ • Leu 1 5 - ceils, or cytotoxic T cells. 7

Table 2. Splenic lymphocyte subpopulations in gastric cancer patients at each stage not given a biological response modifier pre-operatively

Stages I and II III IV ( n = 4 ) ( n = 5 ) ( n = 4 )

Leu-3a+ 3a+ 4+ 4+ 4 - 7+ 7+ 7 - 2a+ 2a+

Leu-8+ 0.6 + 0.31 0.5 + 0.36 1.1 4- 0.53 a 8 - 19.4 +_ 2.74 b 15.4 + 4.53 15.1 + 1.45

HLA-DR+ 7.7 + 2.25 5.6 + 0.67 6.4 + 1.37 HLA-DR- 33.7 _4- 1.26 27.3 + 8.01 30.3 + 2.60 HLA-DR+ 48.8 4- 1.96 51.5 4- 11.55 54.8 + 4.26 L e u - l l - 12.1 4- 3.61 5.5 4- 2.69 b 5.9 + 2.98 a

11+ 4.0 4- 1.22 4.1 + 1.83 2.9 + 1.64 11+ 3.9 4- 2.52 3.9 + 2.13 3.9 + 0.59 15+ 6.1 + 1.09 6.5 4- 3.15 6.6 4- 1.42 15- 14.7 ± 4.65 11.5 + 3.33 12.8 4- 1.92

a: p < 0.10; b: p < 0.05

M. Takahashi et al.: Splenic Lymphocyte Subpopulations 37

Table 3. Comparison of splenic lymphocyte subpopulations between 13 patients with gastric cancer not given a biological response modifier and 5 patients with benign diseases

Gastric cancer Benign disease ( n = 13) ( n = 5)

Leu-3a+. Leu-8+ 3a+. 8 - 4+ HLA-DR+ 4+ H L A - D R - 4 - HLA-DR+ 7+ L e u - l l - 7+ 11+ 7 - 11+ 2a+. 15+ 2a+. 15-

0.7 + 0.50 0.9 + 0.37 16.3 + 3.79 a 12.2 + 4.12

6.5 + 1.69 6.8 + 3.42 30.1 + 5.72 26.6 + 7.55 52.0 + 7.87 58.1 + 8.05

8.2 + 4.30 5.6 + 1.67 3.7 + 1.68 2.4 + 0.93 3.9 + 1.93 3.8 + 0.84 6.4 + 2.20 b 3.9 + 1.04

12.9 + 3.58 11.9 + 4.44

a: p < 0.10; b: p < 0.05

Compar ison with the Splenic L y m p h o c y t e Subpopulat ions o f Patients with Benign Diseases

The ratio of the subpopulations of lymphocytes from the patients who underwent splenectomy for benign diseases is shown in Table 3. There were no differences in the ratios of splenic lymphocyte subpopulations between the gastric cancer and benign disease patients, except for the ratios of helper T and suppressor T cells, which were higher in the gastric cancer patients than in those with a benign disease.

Effects o f the Pre-operat ive Adminis t ra t ion o f B R M

The effects of the pre-operat ive administration of B R M on splenic lymphocytes are given in Table 4. Although pre-operat ive lentinan or OK-432 t reatment led to little alteration in the ratio of subpopulations, compared with findings in the 13 patients not given BRM, the combined administration of lentinan and OK-432 increased the ratio of Leu 4+ • Leu H L A - D R + cells, or

activated T cells, 8 and cytotoxic T cells. With respect to findings in the 8 patients with stage IV gastric cancer, however, activated T and cytotoxic T cells were some- what increased in the 4 patients given BRM, compared with findings in the 4 not given B R M (Table 5).

Discussion

The influence of splenic function on an established carcinoma remains controversial, with both beneficial and detrimental results having been observed. There has also been much discussion concerning the suppress- ive effects of the spleen, and the immunosuppressive effects of spleen cells are experimentally represented by a blocking factor and specific suppressor T cells.l°-14 In contrast, there is documentat ion to support the positive benefits of preservation of the spleen because of the potentiality of regulation ~5 and immunological surveillance. ~6 Thus, bidirectional results have been observed in tumor-bearing animals following splen- ectomy and these phenomena seem to be influenced by the experimental assay, the size of tumor burden, and the timing of splenectomy.17

The effects of splenectomy on gastric cancer patients have only been speculated from survival rates because of the lack of a specific tumor host relationship in gastric cancer. Clinical studies by Sugimachi et al. 18 showed that a non-splenectomized group had a sig- nificantly bet ter survival rate than a splenectomized group. However , Orita et al. 19 and Takahashi et al. 2° suggested that splenectomy resulted in a good prog- nosis. In contrast, Suehiro et al. 21 found that splen- ectomy did not affect the prognosis of patients with gastric cancer.

Since there is no definitive immunologic approach to the tumor-host relation in gastric cancer with respect to the immunologic roles of splenectomy, survival rates

Table 4. Effects of the pre-operative administration of a biological response modifier on splenic lymphocyte subpopulations in gastric cancer patients at stages I-IV

BRM (+)

BRM ( - ) Lentinan OK-432 Lentinan + OK-432 (n = 13) (n = 4) (n = 3) (n = 6)

Leu-3a+ - Leu-8+ 3a+. 8+ 4+ HLA-DR+ 4+ H L A - D R - 4 - HLA-DR+ 7+ Leu- l l+ 7+ 11- 7 - 11+ 2a+. 15+ 2a+- 15-

0.7 +_ 0.5 1.1 _+ 0.31 1.5 +_ 0.29a 1.3 +_ 0.64 a 16.3 _+ 3.7 17.0 + 2.43 18.2 + 2.35 16.1 +_ 2.28 6.5 +_ 1.6 8.0 _ 1.60 14.6 _+ 6.56 11.2 +_ 3.94 a

30.1 + 5.7 28.4 _+ 1.68 30.7 _+ 6.75 31.0 _+ 3.82 52.0 + 7.8 57.0 + 3.47 46.7 + 12.16 48.6 + 4.61 8.2 + 4.3 4.8 + 0.64 11.0 _+ 6.17 6.8 _+ 2.39 3.7 _+ 1.6 3.4 + 2.32 4.7 _+ 2.66 3.6 _+ 1.49 3.9 + 1.9 4.1 +_ 1.46 3.2 + 0.11 5.2 _+ 2.01 6.4 + 2.2 5.8 _+ 1.87 10.4 + 5.86 5.8 + 1.76

12.9 +_ 3.5 12.8 +_ 3.21 15.9 +_ 6.15 18.2 ++_ 3 .25 b

a: p < 0,05; b: p < 0.01

38 M. Takahashi et al.: Splenic Lymphocyte Subpopulations

Table 5. Comparison of splenic lymphocyte subpopulations in stage IV gastric cancer patients

BR~ ( - ) B I ~ (+) (n : 4) (n = 4)

Leu-3a+ o Leu-8+ 1.1 + 0.53 1.3 + 0.64 3a+. 8 - 15.1 _+ 1.45 17.2 + 2.16 4+ • HLA-DR+ 6.4 +_ 1.37 10.5 +_ 3.09 a 4+ • H L A - D R - 30.3 _+ 2.60 31.4 _+ 2.91 4 - • HLA-DR+ 54.8 + 4.26 49.9 + 4.37 7+ • Leu-11- 5.9 _+ 2.98 6.3 _+ 1.95 7+ . 11+ 2.9 + 1.64 2.8 + 0.81 7 - • 11+ 3.9 + 0.59 3.9 _+ 0.22 2a+. 15+ 6.6 _+ 1.42 5.0 + 1.16 2a+. 15- 12.7+_ 1.92 16.7+_ 3.15 ~

a:p <0.10

remain the only point of determination. It is reasonable to assume that splenectomy may somewhat alter the immune state of cancer-bearing patients as the spleen contains 25 per cent of the total lymphatic mass. 22 Thus, we analysed splenic lymphocytes as the first step of assessing the role of the spleen in gastric cancer.

Monoclonal antibodies against T cell surface mem- brane antigens facilitate the division of lymphocytes into serveral subpopulations and provide a constantly reproducible means of quantifying these cells. Two- color flow cytometric analysis opened up the possibility of making a detailed functioning classification of T cells and we used two-color flow cytometry to analyse lymphocytes from the spleen. Falk et alfl 3 examined 25 spleens f rom patients with gastric carcinoma and reported a highly significant reduction of C D 4 + T cells, or helper/suppression inducer T cells. They stated that these results reflect the systemic immunosuppressive and immunodeplet ing effects of a malignant tumor that are probably mediated by tumor-associated cytokines. Toge et al. 24 observed the number of Leu-2+ cells, or suppressor/cytotoxic T cells, in the spleen and draining lymph nodes in gastric cancer patients. They found that both the Leu-2+ cells in the spleen and lymph nodes with metastasis were increased, and suggested possible suppressive effects of the spleen in patients with advanced gastric cancer. Akiyoshi et al. as found that the spleen may be a potential reservoir of the precursor of activated killer ceils in patients with gastric cancer. The reasons for these discrepancies, as Falk et alfl 3 pointed out, may lie in the different histologic features, the tumor stage of the gastric carcinoma studied, and the different methods used. These data were derived from a single color flow cytometric analysis, by which C D 4 + , or Leu 3a+ , T cells include both helper T and suppression inducer T cells, and Leu-2+ cells, both suppressor T and cytotoxic T cells. However , identify- ing each separately is difficult using single color flow cytometry, whereas two-color flow cytometric anal-

ysis is able to differentiate these cells. Thus, a Leu 3a+ • Leu 8+ T cell means a suppression inducer T cell; a Leu 3a+ • Leu 8 - cell means a helper T cell; a Leu 2 a + . Leu 15+ cell means a suppressor T cell; a L e u 2a+ • Leu 15-cell means a cytotoxic T cell and so on.

In the current study, the ratio of suppressor T cells increased in the gastric cancer patients, compared with that in the patients with benign disease. These observations correspond to the findings of Toge et al. 24 in which a single color flow cytometric analysis was used and an increased number of Leu 2a+ cells noted. With respect to the suppression inducer T cells, although Falk et al. 23 reported a marked reduction in these cells in patients wth gastric cancer, the number of Leu 3 a + . Leu 8+ cells in our gastric cancer patients remained unchanged and even increased in the stage IV patients. Presumably, the C D 4 + T cells in Falk 's report were comprised of both suppression inducer and helper T cells, whereas our Leu 3a+ • Leu 8+ T cells were of a single cell group.

In terms of disease progression, splenic NK cells, or Leu 7 + . L e u 1 l - cells, and helper T cells, or Leu 3a+ • Leu 8 - cells, were decreased in proport ion to the stage of the disease. In contrast, the pre-operat ive administration of B R M increased the ratio of activated T cells and cytotoxic T cells. Our results suggest that the immunosuppressive subpopulations located in the spleen increased with the progress of gastric cancer, particularly in the stage IV patients, and that favorable immunological alterations of the subpopulations were induced by the pre-operat ive administration of BRM. Thus, while splenectomy is recommended for stage IV patients, the spleen should probably be preserved in early stage gastric cancer patients.

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