As a link in the series of studies on tumor-specific immunity, in vitro inhibitory effect of sensitized isologous lymph-node cells on the proliferation of C3H
mammary cancer was studied. For this purpose tissue culture was conducted with regional lymph-node cells obtained from truly isologous C3H mouse inoculated with A strain cells derived from C3H mouse mammary cancer along with A cells, and the following results were obtained. In the case of tissue culture with those lymph-node cells obtained from the groups of mice 10 days after the inoculation of 5 X 106 A cells, the inhibitory effect on the proliferation of A cells was most marked, followed by that of those taken on day 14, 7, and 5 decreasing in the order mentioned. In the case with those regional lymph-node cells obtained from mice which did not have recurrence of tumor 1 week after extirpation of 2-week old tumor, the inhibitory effect on proliferation of A cells was marked, with the regional lymph-node cells obtained two weeks after transplantation of 1 × 108 A cells there could be observed no inhibitory effect at all. This suggests that at a certain stage after implantation of such regional lymph- node cells there develops a specific anti-tumor activity in the host.</p

Satoh, Katuaki

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 20, Issue 6 1966 Article 3DECEMBER 1966In vitro studies on tumor-specific immunity byusing C3H mammary cancer-A cells. I.Inhibitory effect of lymph-node cells from thetumor bearing isologous C3H mouse on theproliferation of the tumor cellsKatuaki Satoh∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.In vitro studies on tumor-specific immunity byusing C3H mammary cancer-A cells. I.Inhibitory effect of lymph-node cells from thetumor bearing isologous C3H mouse on theproliferation of the tumor cells∗Katuaki SatohAbstractAs a link in the series of studies on tumor-specific immunity, in vitro inhibitory effect ofsensitized isologous lymph-node cells on the proliferation of C3H mammary cancer was studied.For this purpose tissue culture was conducted with regional lymph-node cells obtained from trulyisologous C3H mouse inoculated with A strain cells derived from C3H mouse mammary canceralong with A cells, and the following results were obtained. In the case of tissue culture withthose lymph-node cells obtained from the groups of mice 10 days after the inoculation of 5 X106 A cells, the inhibitory effect on the proliferation of A cells was most marked, followed bythat of those taken on day 14, 7, and 5 decreasing in the order mentioned. In the case with thoseregional lymph-node cells obtained from mice which did not have recurrence of tumor 1 week afterextirpation of 2-week old tumor, the inhibitory effect on proliferation of A cells was marked, withthe regional lymph-node cells obtained two weeks after transplantation of 1 × 108 A cells therecould be observed no inhibitory effect at all. This suggests that at a certain stage after implantationof such regional lymph- node cells there develops a specific anti-tumor activity in the host.∗PMID: 4227190 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 20, 261-268 (1966)IN VITRO STUDIES ON TUMOR-SPECIFIC IMMUNITYBY USING C3H MAMMARY CANCER-A CELLSI. INHIBITORY EFFECT OF LYMPH-NODE CELLSFROM THE TUMOR BEARING ISOLOGOUS C3HMOUSE ON THE PROLIFERATION OFTHE TUMOR CELLSKatuaki SATOHDepartment of Surgery, Okayama University Medical SchoolOkayama, Japan (Director: Prof. S. Tanaka)Received for publication, November 3, 1966KLEINl established the concept of tumor specific immunity through hisobservation on the primary autochthonous host transplanted with methylcholan-threne-induced sarcoma. Likewise PREHN2, by using dibenz-(a, h)- anthracene-induced tumor, and OLDi , using benzpyrene-induced tumor, demonstrated theexistence of tumor-specific antigen. By using MH 134 cells TAKEDA4 reported thepresence of anti-tumor activity of the lymphoid cells from the spleen of F l strainmice of C3H X dd sensitized with the tumor cells exposed to X-ray by which thetumor cells had lost the activity of cell division. These tumor-specific antigensin vivo have been demonstrated with isologous or autologous animals inoculatedwith viable tumor cells of the same strain after sensitization with previouslytreated tumor cells.For in vitro determination of anti-tumor activity of sensitized lymphoidcells there are experiments by tissue culture method. HARA6, HANAOKAII andROSENAU7 ,8 observed a mutual cell damaging effect between lymphoid cells andtarget cells in such tissue culture, and they demonstrated a direct immune reac-tion between tumor cells and the lymphocytes of the host. However, as they allused homologous lymphocytes and antigenic cells, their experiments are inreality executed homotransplantation immunity in vitro.In the present experiment regional lymph-node cells obtained from C3Hmice sensitized by transplanting mammary cancer derived from the same strainmouse were made to act on target cells, the mammary cancer cells in tissueculture. As the result the existence of anti-tumor activity, the presence of tumorspecific antigen, was demonstrated just as in homotransplantation immunity.Principal findings of the study are described in the following.2611Satoh: In vitro studies on tumor-specific immunity by using C3H mammaryProduced by The Berkeley Electronic Press, 1966262 K. SATOHMATERIALS AND METHODSAnimals: C3H (H-2k ) female mice weighing about 20 g whose genotypewas clear were purchased from The Mouse Colony of Okayama University.They were fed on solid feed. MF of Oriental Yeast Company mixed with somefresh vegetables and grown to the age of 6.......8 weeks old.Sensitization of mice: These 63 animals were divided into 4 groups.To the first group (10 mice) 1 X 108 A strain cells and to the second group (10mice) 5 X 106 cells were inoculated subcutaneously on the back between thescapula, and to the third group (15 mice) 5 X 106 of the same A cells were similarlyinoculated. In the case of groups 1 and 2 regional lymph nodes of axilla wereextirpated two weeks after the inoculation, and lymph nodes in group 3 werefrom 9 mice which did not have reccurrence of tumor 1 week after extirpation of2 week-old tumor. Group 4 was further subdivided into 4 subgroups of 7 animalseach. To each of these subgroups 5 X 106 A strain cells were transplanted in thesame manner as with the other groups, 14, 10, 7 and 5 days respectively beforethe extirpation of the axillary lymph nodes, and the lymph nodes were extir-pated from all the subgroups simultaneously on the same day to be used for theexperiment (Fig. 1).Tumor 1""n,,,r.lftirp.tion !cQ~c.i2"Fig. 1 Graphic Representation of Transplantationof C3H Mammary Cancer CellsTissue culture cells: A-strain cells are the culture cells originally derivedfrom mammary cancer that grew spontaneously in C3H female mice and main-tained in the Pathological Section of Okayama University Cancer Institute andthese cells are first treated with 0.2596 trypsin GKN solution and passed throughthe I50-mesh filter, and cultured in the medium of YLE (Earl's balanced saltsolution containing 0.1 96 (w,Iv) yeast extract and o.596C'lv) lactalbumin hydroly-sate) supplemented with 5096 bovine serum.Lymph-node cell suspension: From six cancer bearing animals each ofthe three groups mentioned above are selected at random and regional lymph2Acta Medica Okayama, Vol. 20 [1966], Iss. 6, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss6/3Tumor Specific Immunity 263nodes are extirpated aseptically while under ether anesthesia. These serve asthe materials for sensitized lymph-node cells of each group. From 15 untreatednormal mice axillary lymph nodes are extirpated in a similar manner and theseserve as the materials for control lymph-node cells. These extirpated lymphnodes are cut into small pieces with ophthalmic scissors and passed through 80-mesh filter. The filtrate is washed with cold Hank's solution by centrifugationat 2, 000 rpm for 5 minutes and these washings are repeated three times. Afterremoval of serum, the rest is suspended in 50% bovine serum plus YLEmedium. Before making suspension in this instance, the countings of viablecells are taken by counting unstained cells by eosin Y staining9 and those show-ing over 80 per cent viable cells are used for the experiment.Culture of lymph-node cells and isologous target cells: For eachgroup, lymph-node cells, A strain cells, and penicillin are mixed in proportionof 16 X 10o/ml, 4 X 104/ml, and 100 r/ml, respectively to make the total volume10 ml. One and half milliliters each of this mixture are pippetted into 6 shorttest tubes and the replicate cell culture is carried out at 37°C by the method ofEVANS et al.lo At the intervals of 24 and 48 hours of incubation, 3 test tubeseach are taken out and the medium is removed by gentle decantation. Then1.5 ml of the crystal violet solution (containing 100 ml distilled water+2.1 gcitric acid +50 mg crystal violet) are added to each of the three test tubes andthe cells are incubated again at 37°C for 30 min. Next, the cells attached tothe wall of test tube are detached gently by a rubber cleaner and by stirringgently a uniform cell suspension is made. A droplet of this suspension is placedon Biirker-Tiirk hemocytometer and the cell counts are taken more than sixtimes for each test tube and the average of three test tubes is taken as thenumber of the increase in A strain cells. The distinction between lymph-nodecells and A strain cells is easy because the former are hyperchromatic and havemuch smaller nucleus while the latter have larger nucleus.RESULTSTwo weeks after the subcutaneous inoculation (on the back and in betweenthe scapula) of A strain cells (derived from mammary cancer of C3H femalemice) there developed tumors of 4. 6 X 3 .5 X 1. 4 cm in average size in the group1 given 1 X 108 cells and 1. 1 X O. 9 X 0 .6 cm in average in the group 2 inoculatedwith 5 X 106 cells. The regional lymph-node cells obtained from the group 1 didnot show any inhibitory effect on A strain cells in culture (Fig. 2); the lymph-node cells from the group 2 did inhibit the proliferation of A cells; those lymph-node cells from group 3 obtained one week after the tumor extirpation, exhibiteda marked inhibitory effect (Fig. 3).3Satoh: In vitro studies on tumor-specific immunity by using C3H mammaryProduced by The Berkeley Electronic Press, 1966264 K. SATOH5 x 10.--;2, 1/;, ,3!J ,.-,~ ,:, ,/; ,/, ,/; ,/-IJ' ", ,~ "~' ~,'24Incubation Time4::Fig. 2 Curves showing the proliferation of A strain cells in the presence ofregional lymph-node cells obtained 2 weeks after inoculation of 1 X108 A cellsNote: 1 denotes control lymph-node cells+A cells, 2 denotes sensitized lymph-nodecells + A cells, and 3 denotes A cells alone.For method, refer to the text.5 x 10'24Incubation Time48Fig. 3 Curves showing inhibitory effect of the regionallymph·node cells obtained2 weeks after transplantation of 5 X 1()6 A cells and the regional lymph-node cellsobtained one week after extirpation of 2-week old tumor, on A strain cellsNote: 1 denotes control lymph-node cells + A strain cells, 2 (5 X106) sensitizedlymph-node cells+A strain cells, 3 (tumor extirpated) sensitized lymph-node cells + A cells, and 4 A strain cells alone.For method, refer to the text.4Acta Medica Okayama, Vol. 20 [1966], Iss. 6, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss6/3Tumor Specific Immunity 265As for group 4 of the regional lymph-node cells extirpated at various inter-vals after the inoculation of 5 X 108 cells, those obtained on the tenth day showedthe most marked inhibitory effect on the growth of A cells in culture, and suchinhibitory effect grew weaker in the regional lymph-node cells in the order ofday 14, 7, and 5 after the inoculation (Fig. 4). Looking at the data in this24Incubation Time48Fig. 4- Curves showing growth-inhibition effect of regional lymph-node cells fromthe animal inoculated with A strain cells on the A strain cells in tissue cultureNote: 1 denotes control lymph-node cells+A strain cells, 2 denotes (14 days later)sensitized lymph-node cells +A cells, 3 denotes (10 days later) sensitized lymph-node cells+A cells, 4 denotes (7 days later) sensitizod lymph-node cells+Acells, 5 denotes (5 days later) sensitized lymph-node ceIls+A cells, and 6denotes A strain cells alone.For the method, refer to the text.series of experiments, it is obvious that, compared with the tissue culture of Astrain cells alone, those A strain cells cultured with untreated normal lymph-node cells invariably tend to accelerate the cell proliferation.DISCUSSIONHIRSCHll demonstrated that the survival time of the inbred mice sensitizedwith spontaneous mammary cancer is prolonged but the animals die of tumorlater. In his experiment with methylcholanthrene-induced sarcoma, KLEIN1 chal-lenged the autochthonous mice, together with groups of isologous animals, pre-treated with irradiated sarcoma cells, increasing doses of viable cells from theoriginal Me-induced sarcoma and found the resistance againt methylcholanthrene-5Satoh: In vitro studies on tumor-specific immunity by using C3H mammaryProduced by The Berkeley Electronic Press, 1966266 K. SATOHinduced sarcoma not only in the primary autochthonous host but also in theisologous mouse. RIGGINS et al. J2 confirmed the immune reaction against spon-taneous mammary cancer of C3H mice, and demonstrated that this reaction isweaker than that of methylcholanthrene-induced sarcoma, and the immunereaction has specificity to immunizing tumor, the extent of which is related tothe inoculating period of tumor cells. In sUl:h a way the existence of tumorspecific antigen in vivo has been made clear. In addition, KLEIN13 demonstratedthat the lymph-node cells of isologous preimmunized mice inhibit the growth ofmethylcholanthrene-induced sarcoma in C57BL strain mice.On the basis of these findings in the present experiments observations ofthe mutual reactions between target cells and sensitized lymph-node cells werecarried out in vitro. As the result it has been clarified that in the regionallymph-node cells obtained from the truly isologous mice to which transplantationof A strain cells derived from C3H mouse was successful there appears anti-tumoractivity at least in a certain stage after the inoculation.While there seems to be no report like the present experiment where theregional lymph-node cells of the host in the case of isograft transplantation showanti-tumor activity to antigenic tumor cells, ROSENAU7•8 reported that lymph-nodecells obtained from BALB/c strain mouse sensitized with L cells derived fromC3H mouse in the absence of complement adhered specifically to L-cells and theseL-cells were gradually damaged. HARAr., in his mixed cell tissue culture oflTC-II strain cells (derived from Ehrlich tumor) and the corresponding sensitizedlymph-node cells, found that the growth of lTC-ll cells was inhibited. BRONDZ14showed that immune mouse lymphocytes exhibit cytotoxic effect on Sal cells.Everyone of these shows the action behaviors of antigenic cells at homotrans-plantation, and the action beheviors of sensitized lymphoid cells in vitro seem torepresent the action of the host lymph-node cells against excess histocompatibilityantigen of homologous transplant in vivo.From the fact that the skin graft transplantation between C3H mouse (usedin this experiment) survived permanently in place and there could be recongnizedno immunological change at alp6, it is assumed that C3H mouse is isologous asfar as histocompatibility gene is concerned. Consequently, it might be fairlyreasonable to assume that the anti-tumor activity by the regional lymph node to·mammary cancer derived from isologous mouse is a reflection of immunologicalreaction in vitro of lymph-node cells to mammary cancer cell specific antigenother than histocompatibility gene itself.Those regional lymph-node cells from the group extirpated of tumor showmuch stronger inhibitory action than the tumor bearing group, and those lymph-node cells from the group of mice on the threshold of death from tumors do notshow any inhibitory action.6Acta Medica Okayama, Vol. 20 [1966], Iss. 6, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss6/3Tumor Specific Immunity 267This fact seems to indicate that the lymph-node cells in the terminal stageof cancer become paralytic because of an enormous amount of antigens fromtumor cells and react as a sort of enhancement factor to tumor. In either case,these two factors seem to be involved in the unlimited growth of cancer.SUMMARYAs a link in the series of studies on tumor-specific immunity, in vitro inhi-bitory effect of sensitized isologous lymph-node cells on the proliferation of C3Hmammary cancer was studied. For this purpose tissue culture was conductedwith regional lymph-node cells obtained from truly isologous C3H mouse inocu-lated with A strain cells derived from C3H mouse mammary cancer along withA cells, and the following results were obtained.In the case of tissue culture with those lymph-node cells obtained from thegroups of mice 10 days after the inoculation of 5 X 106 A cells, the inhibitoryeffect on the proliferation of A cells was most'marked, followed by that of thosetaken on day 14, 7, and 5 decreasing in the order mentioned.In the case with those regional lymph-node cells obtained from mice whichdid not have recurrence of tumor 1 week after extirpation of 2-week old tumor,the inhibitory effect on proliferation of A cells was marked, with the regionallymph-node cells obtained two weeks after transplantation of 1 X 108 A cellsthere could be observed no inhibitory effect at all. This suggests that at acertain stage after implantation of such regional lymph-node cells there developsa specific anti-tumor activity in the host.ACKNOWLEDGEMENTThe author wishes to express profound thanks to Professor Sanae TANAKA of our Departmentfor his painstaking proof reading, and to Professor Jiro SATOH of Pathology Section of CancerInstitute, Okayama University Medical School and Dr. Kunzo ORITA of our Department for theirkind guidance throughout this work.REFERENCES1. KLEIN, G. et al.: Demonstration of resistance against methycholanthrene-induced sarcomain the primary autochthonous host. Cancer Res. 20, 1561, 19602. PREHN, R. T.: Tumor-specific immunity to transplanted dibenz- (a, h)-anthracene-inducedsarcoma. Cancer Res. 20, 1614, 19603. OLD, L. H. et al.: The role of the retkuloendothelial system in the host reaction to neoplasia.Cancer Res. 21, 1281, 19614. TAKEDA, K.: Immunogenic study in antitumor antibody production against transplantabletumors. Okayama Igakkai Zassi 75, 103, 1963 (in Japanese)5. HARA, S.: Cellular antibody in mice bearing Ehrlich cancer. 1. A quantitative study onantitumor activity of cellular antibody in vitro. Acta Med. Okayama 19, 91, 19657Satoh: In vitro studies on tumor-specific immunity by using C3H mammaryProduced by The Berkeley Electronic Press, 1966268 K. SATOH6. HANAOKA, M. and NOTAKE, K.: Quantitative studies on the cellular antibody in vitro. I.Inhibitory effect of sensitized homologous lymph node cells on strain SCI of cultured leukemiccells. Ann. Reb. Inst. Virus., Res., Kyoto Univ. 5. 134, 19627. ROSENAU, W. and MOON, H. D.: Lysis of homologous cells by sensitized lymphocytes intissue culture. ]. Nat. Cancer Inst. 27, 471. 19618. ROSENAU, W.: Interaction of lymphoid cells with target cells in tissue culture. Cell-BoundAntibodies. ed. by B. Amos and J. H. Koprowski, p. 75, Wister Inst. Press, Philadelphia,19639. SCHREK, R.: A methOd of counting the viable cells in normal and in malignant cell sus-pensions. Am. ]. Cancer 28, 389, 193610. EVANS, V. J., EARLE, W. R., SANFORD, K. K., SHANNON, J. E. and WALTZ, H. K.: Thepreparation and handling of replicate tissue cultures for quantitative studies. ]. Nat. CancerInst. 11, 907, 195111. HIRSCH, M. H. et al.: Can the inbred mouse be immunized against its own tumor? CancerRes. 18, 244, 195812. RIGGINS, R. S. ann PILCH, Y. H.: Immunity to spontaneous and methylcholanthrene-inducedtumors in inbred mice. Cancer Res. 24, 1994, 196413. KLEIN, E. and SJOGREN, H. 0.: Humoral and cellular factors in homograft and isograft im-munity against sarcoma cells. Cancer Res. 20, 452. 196014. BRONDZ, B. D.: Interaction of immune lymphocytes in vitro with normal and neoplastic tissuecells. Folia Bwl. 10, 164, 196415. KOKUMAI, Y.: Acta Med. Okayama in press8Acta Medica Okayama, Vol. 20 [1966], Iss. 6, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss6/3

In vitro studies on tumor-specific immunity by using C3H mammary cancer-A cells. I. Inhibitory effect of lymph-node cells from the tumor bearing isologous C3H mouse on the proliferation of the tumor cells

In vitro studies on tumor-specific immunity by using C3H mammary cancer-A cells. I. Inhibitory effect of lymph-node cells from the tumor bearing isologous C3H mouse on the proliferation of the tumor cells

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