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Surface Markers of Lymphocytes Infiltrating Seminoma Tissue
0022-5347 /80/1246-0827$02.00/0 Vol. 124, December Printed in U.S.A..
THE JOURNAL OF UROLOGY
Copyright© 1980 by The Williams & Wilkins Co.
SURFACE MARKERS OF LYMPHOCYTES INFILTRATING SEMINOMA TISSUE HIDEYUKI AKAZA, * KATSpMI KOBAYASHI, T AKASHI UMEDA
AND
T ADAO NIIJIMA
From the Department of Urology, Facul~y of M~edicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
ABSTRACT
Infiltrating ly-:rnphocytes were separated from human seminoma tissue obtained surgically. The :relative distributions of B and T lymphocytes, and a subset of T lymphocytes with IgG-Fc receptors were measured by the rosette-forming method in peripheral blood and in seminoma tissue from 7 patients with pure seminoma of the testis. The proportions of T and T G cells were increased in seminoma tissue compared to those of peripheral blood from the same patients, whereas the proportion of B cells did not change. This :result might correlate with the favorable prngnosi.s of seminoma usually accompanied with lymphocytic infiltration. Seminoma h.as the most favorable prognosis of aH germ cell tumors of the testis. Varying amounts of lymphocytic infiltrations are observed in almost all seminomas and granulative reactions are observed in about half the cases. 1 Dixon and Moore reported that these types of stromal reactions were associated with a good prognosis for seminoma. 2 This fact may suggest the possibility of an immune response against seminoma cells from tumor-bearing patients. However, there have been little direct studies on the tumor-host reaction within seminoma tissue. The relative distributions of B and T cells, and a subset of T cells with IgG-Fc receptors (Tc) were studied in seminoma tissue, chronic orchitis tissue and normal testes from patients with prostatic cancer in comparison to those of peripheral blood from the same patient. PATIENTS
We studied 7 patients with pure seminoma of the testis with an average age of 45 years and a range of 41 to 60 years, 3 patients with chronic orchitis with an average age of 43 years and a range of 40 to 49 years and 3 patients with prostatic cancer with an average age of 68 years and a range of 54 to 78 years. All seminomas were TlNOMO according to the classification of the International Union Against Cancer. 3 METHOD
Separation of lymphocytes from tissue. Specimens were obtained at orchiectomy and normal testes were obtained at castration from patients with prostatic cancer. After obvious necrotic areas were cut away and discarded the specimens were in RPMI-1640 medium (pH 7.4), washed and minced to l mm. in maximum dimension using scissors without any enzyme at room temperature. Single cells were squeezed out gentle pressure between 2 slide glasses. The cell suspension was passed through 3 thicknesses of gauze so that the fragments of specimen were eliminated. The suspended cells were washed 3 times centrifugation at 100 times gravity for 5 minutes with RPMI-1640 medium and incubated with carbonyl iron for depletion of phagocytic cells. 4 Erythrocytes in the starting suspension were mixed scarcely compared to other cell components. Therefore, the degree of contamination of the suspension by peripheral lymphocyte was thought to be negligible. Lymphocytes were separated from the suspension on ficollhypaque gradient by centrifugation at 400 times gravity for 30 minutes at 4C. The proportion of macrophage in the cell susµeu,;1.uu was <3 per cent as tested by peroxidase exclusion.
From 65 to 70 per cent of the dispersed cells were lymphocytes in cases of seminoma and chronic orchitis. The absolute number of the lymphocytes from l gm. tissue was related mainly to the degree of microscopic lymphocytic infiltration except the cases of normal testes from patients with prostatic cancer who had no lymphocytic infiltration. Approximately 107 lymphocytes were recovered from 1 gm. seminoma tissue in case 6 and 104 lymphocytes were recovered from l gm. chronic orchitis tissue in case 10. Lymphocyte in peripheral blood. The method used for phocyte separation and purification was according to that of Tebbi, with carbonyl iron for depletion of phagocytic cells and with ficoll-hypaque gradient for lymphocyte isolation. 4 Peripheral blood was obtained within 1 hour of orchiectomy or cast:ra-, tion. Enumeration of lymphocyte subpopulations. E-rosette assays for T lymphocytes were performed according to the method of Wybran and Fudenberg, 5 complement receptor-bearing cells (B cells) were assayed by the method of Raben and associates,° and T G cells were assayed according to the slightly modified method of Gupta and Good. 7 Macrophage-depleted lymphocytes were rosetted wi.th neuraminidase-treated sheep rocytes for T cell assay and with sheep erythrocytes coated with IgM antibody and human complements of AB blood type donor for the assay of complement receptors bearing lympho-cyte, respectively. T cells (E-rosette) were separated from non-Teel.ls (non-Erosette) on ficoll-hypaque gradient. Sheep erythrocytes attached to the lymphocytes were lysed with distilled water. Purified T lymphocytes were cultured in RPMI-1640 medium containing 10 per cent heat-inactivated fetal calf serum. Then, 0.1 ml. purified T lymphocyte suspension (2 X 106 /ml.) was mixed with 0.1 ml. IgG coated ox erythrocytes suspension, centrifuged at 100 times gravity for 5 minutes and incubated at 4C for l hour. The pellet was resuspended gently and 200 lymphocytes were counted on a hemocytometer under a microscope. A lymphocyte attached by ~3 erythrocytes was considered a rosette. T O cells were expressed in terms of percentage of the rosetting cells in the total T cell population. ·
Accepted for publication March 7, 1980. * Requests for reprints: Department of Urology, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. 827
RESULTS
Complete data on all materials tested are shown in table l. In cases of testicular seminoma T and To cells were increased significantly compared to those of peripheral blood from the same patients (p <0.001). However, when they were compared to those of peripheral lymphocytes from 12 normal controls only To cells were increased significantly (p <0.001) (table In chronic orchitis cases no such results were noted.
828
AKAZA AND ASSOCIATES TABLE
1. Relative distributions of lymphocyte subpopulations (%) Tissue Infiltrating Lymphocyte
Peripheral Lymphocyte
Material Pt. Age
TCell
Tc Cell*
B Cell
TCell
Tc Cell
B Cell
42 42 41 42 43 42 60
76 70 68 61 65 73 54
29 30 32 18 31 21
9 14 9 11 12 13 15
50 69 64 47 49 63 48
8 14 8 12 9 7 12
49 40 40
67 ± 6.9 40 37 49 42 ± 5.0
25 ± 6.2 12 9 10 10 ± 1.2
12 ± 2.2 25 20 19 21 ± 2.6
56 ± 8.6 60 65 70 65 ± 4.1 53 60 47 53 ± 5.3
10 ± 2.4 13 12 6 10 ± 3.1 10 13
10 12 11 12 10 11 11 11 ± 0.8 13 17 16 15 ± 1.7 17 18 9 15 ± 4.0
Seminoma Seminoma Seminoma
Seminoma Seminoma Seminoma
Seminoma Mean ± standard deviation Chronic orchitis Chronic orchitis Chronic orchitis Mean ± standard deviation
54 72 78
Normal testis (prostatic Ca)
16
No lymphocyte obtained
Mean ± standard deviation
18
14 ± 3.3
* Proportion of Tc cell in total T cell. TABLE
2. Subpopulation of 12 normal peripheral blood lymphocyte
(mean percentage ± standard deviation) T Cell 63 ± 8.0
Tc Cell 9
± 4.0
B Cell 15 ± 7.0
peripheral lymphocyte after contact with tumor antigens within seminoma tissue or other? What are the functional capabilities of infiltrating T and T G cells? Do they have any relationship to the good prognosis of seminoma? These problems are left to be studied.
DISCUSSION
REFERENCES
Recent studies have indicated that some populations of human peripheral lymphocytes have antitumor activity. 8 Of the germ cell tumors only seminomas have abundant lymphocytic infiltration in the tissue and this fact has been considered to be one of reasons for its favorable prognosis. 2 It is an interesting and fundamental problem to clarify the lymphocyte subpopulations that infiltrate seminoma tissue and that may have a possible role in tumor-host reaction. The indirect immunofluorescence method with antithymus-antiserum9 may be used for the detection of T cells within tissue but it cannot be used for the detection of TG cells. Our study indicates that the proportions of T and TG cells were increased in seminoma tissue compared to those of peripheral lymphocytes from the same patients. These results were not the same in cases of chronic orchitis. Since normal testes from patients with prostatic cancer had no available amount of lymphocytic infiltration they could not be used as controls in this study. The increments of the proportion of T cells in seminoma tissue may be compatible with the least metastatic chance of all germ cell malignancies of the testis because T cells are postulated to have the most important role in cell-mediated cytotoxicity. 9 • 10 According to Moretta and associates T G cells have a suppressive effect against immunoglobulin production by B cells 11 and according to Gupta and associates they demonstrate natural killer activity and antibody-dependent cellular cytotoxic activity.8 Dennert and Hatlen have shown that T cell-mediated cytotoxicity might be enhanced by the presence of tumor antigens.12 Several questions have arisen from our results relative to the regional tumor-host reaction manifested by the infiltrating lymphocytes in the tumor. What makes the different proportions of lymphocyte subpopulations between seminoma tissue and peripheral blood from the same patient? Is it accumulation of the peripheral component or further differentiation of
1. Mostofi, F. K. and Price, E. B.: Tumors of the male genital system. In: Atlas of Tumor Pathology. Washington, D. C.: Armed Forces Institute of Pathology, 2nd series, fasc. 8, p. 21, 1973. 2. Dixon, F. J. and Moore, R. A.: Testicular tumors; a clinicopathological study. Cancer, 6: 427, 1953. 3. Harmer, M. H.: TNM classification of malignant tumors. International Union Against Cancer, 3rd ed., p. 122, 1978. 4. Tebbi, K.: Purification of lymphocytes. Lancet, 1: 1392, 1973. 5. Wybran, J. and Fudenberg, H. H.: Thymus-derived rosette-forming cells in various human disease states: cancer, lymphoma, bacterial and viral infections, and other diseases. J. Clin. Invest., 52: 1026, 1973. 6. Raben, M., Walach, N., Galili, U. and Schlesinger, M.: The effect of radiation therapy on lymphocyte subpopulations in cancer patients. Cancer, 37: 1417, 1976. 7. Gupta, S. and Good, R. A.: Subpopulations of human T lymphocytes. V. T lymphocytes with receptors for immunoglobulin Mor G in patients with primary immunodeficiency disorders. Clin. Immunol. Immunopathol., 11: 292, 1978. 8. Gupta, S., Fernandes, G., Nair, M. and Good, R. A.: Spontaneous and antibody-dependent cell-mediated cytotoxicity by human T cell subpopulations (killer cells/ effector T cells/immunoglobulin receptor/T cell subsets). Proc. Natl. Acad. Sci., 75: 5137, 1978. 9. Husby, G., Hoagland, P. M., Strickland, R. G. and Williams, R. C., Jr.: Tissue T and B cell infiltration of primary and metastatic cancer. J. Clin. Invest., 57: 1471, 1977. 10. Sanderson, C. J. and Glauert, A. M.: The mechanism of T-cell mediated cytotoxicity. VI. T-cell projections and their role in target cell killing. Immunology, 36: 119, 1979. 11. Moretta, L., Webb, S. R., Grossi, C. E., Lydyard, P. M. and Cooper, M. D.: Functional analysis of two human T-cell subpopulations: help and suppression of B-cell responses by T cells bearing receptors for IgM or IgG. J. Exp. Med., 146: 184, 1977. 12. Dennert, G. and Hatlen, L. E.: Induction and properties of cytotoxic T cells specific for hapten-coupled tumor cells. J. Immunol., 114: 1705, 1975.
THE JOURNAL OF UROLOGY
Copyright© 1980 by The Williams & Wilkins Co.
SURFACE MARKERS OF LYMPHOCYTES INFILTRATING SEMINOMA TISSUE HIDEYUKI AKAZA, * KATSpMI KOBAYASHI, T AKASHI UMEDA
AND
T ADAO NIIJIMA
From the Department of Urology, Facul~y of M~edicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
ABSTRACT
Infiltrating ly-:rnphocytes were separated from human seminoma tissue obtained surgically. The :relative distributions of B and T lymphocytes, and a subset of T lymphocytes with IgG-Fc receptors were measured by the rosette-forming method in peripheral blood and in seminoma tissue from 7 patients with pure seminoma of the testis. The proportions of T and T G cells were increased in seminoma tissue compared to those of peripheral blood from the same patients, whereas the proportion of B cells did not change. This :result might correlate with the favorable prngnosi.s of seminoma usually accompanied with lymphocytic infiltration. Seminoma h.as the most favorable prognosis of aH germ cell tumors of the testis. Varying amounts of lymphocytic infiltrations are observed in almost all seminomas and granulative reactions are observed in about half the cases. 1 Dixon and Moore reported that these types of stromal reactions were associated with a good prognosis for seminoma. 2 This fact may suggest the possibility of an immune response against seminoma cells from tumor-bearing patients. However, there have been little direct studies on the tumor-host reaction within seminoma tissue. The relative distributions of B and T cells, and a subset of T cells with IgG-Fc receptors (Tc) were studied in seminoma tissue, chronic orchitis tissue and normal testes from patients with prostatic cancer in comparison to those of peripheral blood from the same patient. PATIENTS
We studied 7 patients with pure seminoma of the testis with an average age of 45 years and a range of 41 to 60 years, 3 patients with chronic orchitis with an average age of 43 years and a range of 40 to 49 years and 3 patients with prostatic cancer with an average age of 68 years and a range of 54 to 78 years. All seminomas were TlNOMO according to the classification of the International Union Against Cancer. 3 METHOD
Separation of lymphocytes from tissue. Specimens were obtained at orchiectomy and normal testes were obtained at castration from patients with prostatic cancer. After obvious necrotic areas were cut away and discarded the specimens were in RPMI-1640 medium (pH 7.4), washed and minced to l mm. in maximum dimension using scissors without any enzyme at room temperature. Single cells were squeezed out gentle pressure between 2 slide glasses. The cell suspension was passed through 3 thicknesses of gauze so that the fragments of specimen were eliminated. The suspended cells were washed 3 times centrifugation at 100 times gravity for 5 minutes with RPMI-1640 medium and incubated with carbonyl iron for depletion of phagocytic cells. 4 Erythrocytes in the starting suspension were mixed scarcely compared to other cell components. Therefore, the degree of contamination of the suspension by peripheral lymphocyte was thought to be negligible. Lymphocytes were separated from the suspension on ficollhypaque gradient by centrifugation at 400 times gravity for 30 minutes at 4C. The proportion of macrophage in the cell susµeu,;1.uu was <3 per cent as tested by peroxidase exclusion.
From 65 to 70 per cent of the dispersed cells were lymphocytes in cases of seminoma and chronic orchitis. The absolute number of the lymphocytes from l gm. tissue was related mainly to the degree of microscopic lymphocytic infiltration except the cases of normal testes from patients with prostatic cancer who had no lymphocytic infiltration. Approximately 107 lymphocytes were recovered from 1 gm. seminoma tissue in case 6 and 104 lymphocytes were recovered from l gm. chronic orchitis tissue in case 10. Lymphocyte in peripheral blood. The method used for phocyte separation and purification was according to that of Tebbi, with carbonyl iron for depletion of phagocytic cells and with ficoll-hypaque gradient for lymphocyte isolation. 4 Peripheral blood was obtained within 1 hour of orchiectomy or cast:ra-, tion. Enumeration of lymphocyte subpopulations. E-rosette assays for T lymphocytes were performed according to the method of Wybran and Fudenberg, 5 complement receptor-bearing cells (B cells) were assayed by the method of Raben and associates,° and T G cells were assayed according to the slightly modified method of Gupta and Good. 7 Macrophage-depleted lymphocytes were rosetted wi.th neuraminidase-treated sheep rocytes for T cell assay and with sheep erythrocytes coated with IgM antibody and human complements of AB blood type donor for the assay of complement receptors bearing lympho-cyte, respectively. T cells (E-rosette) were separated from non-Teel.ls (non-Erosette) on ficoll-hypaque gradient. Sheep erythrocytes attached to the lymphocytes were lysed with distilled water. Purified T lymphocytes were cultured in RPMI-1640 medium containing 10 per cent heat-inactivated fetal calf serum. Then, 0.1 ml. purified T lymphocyte suspension (2 X 106 /ml.) was mixed with 0.1 ml. IgG coated ox erythrocytes suspension, centrifuged at 100 times gravity for 5 minutes and incubated at 4C for l hour. The pellet was resuspended gently and 200 lymphocytes were counted on a hemocytometer under a microscope. A lymphocyte attached by ~3 erythrocytes was considered a rosette. T O cells were expressed in terms of percentage of the rosetting cells in the total T cell population. ·
Accepted for publication March 7, 1980. * Requests for reprints: Department of Urology, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. 827
RESULTS
Complete data on all materials tested are shown in table l. In cases of testicular seminoma T and To cells were increased significantly compared to those of peripheral blood from the same patients (p <0.001). However, when they were compared to those of peripheral lymphocytes from 12 normal controls only To cells were increased significantly (p <0.001) (table In chronic orchitis cases no such results were noted.
828
AKAZA AND ASSOCIATES TABLE
1. Relative distributions of lymphocyte subpopulations (%) Tissue Infiltrating Lymphocyte
Peripheral Lymphocyte
Material Pt. Age
TCell
Tc Cell*
B Cell
TCell
Tc Cell
B Cell
42 42 41 42 43 42 60
76 70 68 61 65 73 54
29 30 32 18 31 21
9 14 9 11 12 13 15
50 69 64 47 49 63 48
8 14 8 12 9 7 12
49 40 40
67 ± 6.9 40 37 49 42 ± 5.0
25 ± 6.2 12 9 10 10 ± 1.2
12 ± 2.2 25 20 19 21 ± 2.6
56 ± 8.6 60 65 70 65 ± 4.1 53 60 47 53 ± 5.3
10 ± 2.4 13 12 6 10 ± 3.1 10 13
10 12 11 12 10 11 11 11 ± 0.8 13 17 16 15 ± 1.7 17 18 9 15 ± 4.0
Seminoma Seminoma Seminoma
Seminoma Seminoma Seminoma
Seminoma Mean ± standard deviation Chronic orchitis Chronic orchitis Chronic orchitis Mean ± standard deviation
54 72 78
Normal testis (prostatic Ca)
16
No lymphocyte obtained
Mean ± standard deviation
18
14 ± 3.3
* Proportion of Tc cell in total T cell. TABLE
2. Subpopulation of 12 normal peripheral blood lymphocyte
(mean percentage ± standard deviation) T Cell 63 ± 8.0
Tc Cell 9
± 4.0
B Cell 15 ± 7.0
peripheral lymphocyte after contact with tumor antigens within seminoma tissue or other? What are the functional capabilities of infiltrating T and T G cells? Do they have any relationship to the good prognosis of seminoma? These problems are left to be studied.
DISCUSSION
REFERENCES
Recent studies have indicated that some populations of human peripheral lymphocytes have antitumor activity. 8 Of the germ cell tumors only seminomas have abundant lymphocytic infiltration in the tissue and this fact has been considered to be one of reasons for its favorable prognosis. 2 It is an interesting and fundamental problem to clarify the lymphocyte subpopulations that infiltrate seminoma tissue and that may have a possible role in tumor-host reaction. The indirect immunofluorescence method with antithymus-antiserum9 may be used for the detection of T cells within tissue but it cannot be used for the detection of TG cells. Our study indicates that the proportions of T and TG cells were increased in seminoma tissue compared to those of peripheral lymphocytes from the same patients. These results were not the same in cases of chronic orchitis. Since normal testes from patients with prostatic cancer had no available amount of lymphocytic infiltration they could not be used as controls in this study. The increments of the proportion of T cells in seminoma tissue may be compatible with the least metastatic chance of all germ cell malignancies of the testis because T cells are postulated to have the most important role in cell-mediated cytotoxicity. 9 • 10 According to Moretta and associates T G cells have a suppressive effect against immunoglobulin production by B cells 11 and according to Gupta and associates they demonstrate natural killer activity and antibody-dependent cellular cytotoxic activity.8 Dennert and Hatlen have shown that T cell-mediated cytotoxicity might be enhanced by the presence of tumor antigens.12 Several questions have arisen from our results relative to the regional tumor-host reaction manifested by the infiltrating lymphocytes in the tumor. What makes the different proportions of lymphocyte subpopulations between seminoma tissue and peripheral blood from the same patient? Is it accumulation of the peripheral component or further differentiation of
1. Mostofi, F. K. and Price, E. B.: Tumors of the male genital system. In: Atlas of Tumor Pathology. Washington, D. C.: Armed Forces Institute of Pathology, 2nd series, fasc. 8, p. 21, 1973. 2. Dixon, F. J. and Moore, R. A.: Testicular tumors; a clinicopathological study. Cancer, 6: 427, 1953. 3. Harmer, M. H.: TNM classification of malignant tumors. International Union Against Cancer, 3rd ed., p. 122, 1978. 4. Tebbi, K.: Purification of lymphocytes. Lancet, 1: 1392, 1973. 5. Wybran, J. and Fudenberg, H. H.: Thymus-derived rosette-forming cells in various human disease states: cancer, lymphoma, bacterial and viral infections, and other diseases. J. Clin. Invest., 52: 1026, 1973. 6. Raben, M., Walach, N., Galili, U. and Schlesinger, M.: The effect of radiation therapy on lymphocyte subpopulations in cancer patients. Cancer, 37: 1417, 1976. 7. Gupta, S. and Good, R. A.: Subpopulations of human T lymphocytes. V. T lymphocytes with receptors for immunoglobulin Mor G in patients with primary immunodeficiency disorders. Clin. Immunol. Immunopathol., 11: 292, 1978. 8. Gupta, S., Fernandes, G., Nair, M. and Good, R. A.: Spontaneous and antibody-dependent cell-mediated cytotoxicity by human T cell subpopulations (killer cells/ effector T cells/immunoglobulin receptor/T cell subsets). Proc. Natl. Acad. Sci., 75: 5137, 1978. 9. Husby, G., Hoagland, P. M., Strickland, R. G. and Williams, R. C., Jr.: Tissue T and B cell infiltration of primary and metastatic cancer. J. Clin. Invest., 57: 1471, 1977. 10. Sanderson, C. J. and Glauert, A. M.: The mechanism of T-cell mediated cytotoxicity. VI. T-cell projections and their role in target cell killing. Immunology, 36: 119, 1979. 11. Moretta, L., Webb, S. R., Grossi, C. E., Lydyard, P. M. and Cooper, M. D.: Functional analysis of two human T-cell subpopulations: help and suppression of B-cell responses by T cells bearing receptors for IgM or IgG. J. Exp. Med., 146: 184, 1977. 12. Dennert, G. and Hatlen, L. E.: Induction and properties of cytotoxic T cells specific for hapten-coupled tumor cells. J. Immunol., 114: 1705, 1975.