Effects of isolated tumor lymphocytes alone and with adherent cells

Summary The effect on the growth of gradient-isolated mouse mammary tumor cells of different populations of lymphoid cells were evaluated in micrototoxicity assays. Variable effects were obtained with tumor-bearer lymph node and spleen cells: in some experiments growth stimulation occurred, whereas in others inhibition was observed. Mixed effector populations gave more regular results: adherent spleen cells added to lymph node or spleen lymphocytes inhibited tumor cell growth in six of nine experiments; inhibition occurred when either of the effector populations in the mixture was derived from the tumor-bearing mouse. Tumor-associated lymphoid cells (TAL) stimulated growth of the tumor cells in five of seven experiments. However, TAL inhibited tumor growth when combined with adherent spleen cells from tumor-bearing animals. In contrast with the peripheral lymphoid cells, admixture of control adherent cells from normal animals with TAL did not inhibit growth. No natural killer effect was seen in these growth inhibition assays. These data indicate that lymphoid populations capable of inhibiting tumor cell growth can be found in tumor-bearing animals, but such combination of active cells are not present at the tumor site.     

Immunogenicity of viable tumor cells

Summary The immunogenicity of KMT-17 fibrosarcoma cells which had been xenogenized by infection with FV was compared to that of KMT-17 cells which had been admixed with BCG. We report here that 10⁵ and 10⁶ KMT-17 cells also grew progressively to kill rats, but when 10⁵ KMT-17 cells were administered with BCG the tumor cells did not grow in the majority of rats. The strength of immunogenicity (ETD₅₀), as measured by the number of immunizing cells required for a suppression of growth of 10⁷ KMT-17 cells in 50% of the rats, was 2.1×10³ for FV-KMT-17 and 36.3×10³ for BCG+KMT-17. The tumor cell dose (LTD₅₀) which was required to kill 50% of the rats immunized with 10⁵ FV-KMT-17 was more than 10,000 times higher than that found in normal rats, whereas the number of tumor cells required to kill 50% of the rats immunized with the same number of BCG+KMT-17 was only 3,680 times higher than the amount found in normal rats. Thus the immunogenicity of FV-KMT-17 is much stronger than that of BCG+KMT-17.The difference in immunogenicity between the two vaccines was also observed in the tumor-neutralizing activities of spleen cells obtained from rats which had been immunized with both vaccines, as measured by a Winn assay. Moreover, the antitumor activity of spleen cells from rats immunized with FV-KMT-17 was concentrated in the carrageenan-resistant and plastic nonadherent cells, while that of spleen cells from rats immunized with BCG+KMT-17 was observed in carrageenan-sensitive and plastic adherent cells as well as in nonadherent cells. The involvement of different effector cells indicates that different mechanisms operate in the antitumor resistance in rats immunized with either FV-KMT-17 or BCG+KMT-17. Abbreviations used: FV, Friend leukemia virus; FV-KMT-17, Friend leukemia virus infected KMT-17 cells; EDT₅₀, a 50% effective tumor dose; LTD₅₀, a 50% lethal tumor dose     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

Enhanced susceptibility of target KMT-17 cells to activated macrophages by treatment with the antitumor agent bleomycin

Summary We observed that after KMT-17 cells had been treated with bleomycin (BLM), even with a dose as high as 160 g/ml, they were still able to form colonies in soft agar. We then studied the susceptibility of KMT-17 cells treated with BLM to activated macrophages. During a colony inhibition assay, BLM-treated KMT-17 cells were found to be much more susceptile to activated macrophages than nontreated KMT-17 cells, moreover, a tumor neutralizing assay showed that the growth of BLM-treated KMT-17 cells was also significantly inhibited by activated macrophages as compared with nontreated KMT-17 cells. Macrophages activated by both BLM and the Nocardia rubra cell wall skeleton were able to mediate such tumor inhibition activity in BLM-treated KMT-17 cells. Activated macrophages did not seem to have strong antitumor activity against nontreated KMT-17 cells in vivo, however, the life span of the rats which were inoculated i. p. with KMT-17 cells was significantly expanded after the tumorbearing rats were given BLM i.p. The data presented here suggest that not only does BLM have a direct tumoricidal effect on KMT-17 cells, it also regulates immunosensitivity of targets to immune effectors. We also discuss the mechanism for enhancing the susceptibility of KMT-17 cells to activated macrophages brought about by treatment with BLM.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education, Science, and Culture     

Increased or decreased immunogenicity of tumor-associated antigen according to the amount of virus-associated antigen in rat tumor cells infected with friend virus

Summary The immunogenicity of tumor-associated antigen (TAA) in 3-methylcholanthrene-induced rat fibrosarcoma KMT-17 cells was investigated with particular reference to the amount of virus-associated antigen (VAA) produced on the cell surface after artificial infection of tumor cells with Friend murine leukemia virus (FV). To compare the TAA immunogenicity of FV-infected KMT-17 tumor cells (FV-KMT-17) with non-infected KMT-17 cells, rats were immunized with the above two tumor-line cells, and the growth of original non-infected KMT-17 cells was observed. The result showed that TAA immunogenicity was not always paralleled by the amount of VAA newly produced. The amount of VAA increased in proportion to the number of passage generations of FV-KMT-17 cells through FV-tolerant rats that had been neonatally injected with Friend virus. The TAA immunogenicity of FV-KMT-17 cells after two passages was lower than that of non-infected KMT-17 cells. High TAA immunogenicity was observed in FV-KMT-17 tumor cells of the fourth passage generation; it then weakened gradually after subsequent passages of FV-KMT-17 tumor cells, and finally dropped to a level lower than the immunogenicity of the original non-infected tumor cells. However, such variations of immunogenicity were never observed in FV-tolerant rats. These observations suggest that TAA immunogenicity definitely increases when an appropriate amount of VAA is expressed on the tumor cell surface and decreases when a relatively low or excessive amount of VAA is expressed. We discuss the mechanisms leading to the above findings. Abbreviations used in this paper are: FV, Friend murine leukemia virus; TAA, KMT-17 tumor-associated antigen; VAA, FV-associated antigen     

The mechanism of tumor growth inhibition by tumor-specific Lyt-1+2-T cells. I. Antitumor effect of Lyt-1+2-T cells depends on the existence of adherent cells

In the present study we establish an assay system of tumor growth inhibition with the use of a diffusion chamber and investigate the mechanism by which tumor-specific Lyt-1+2-T cells exhibit their inhibiting effect on tumor cell growth. When a diffusion chamber containing X5563 plasmacytoma cells together with normal syngeneic C3H/HeN spleen cells was implanted in the peritoneal cavity of C3H/HeN mice, these tumor cells continued to proliferate at least 7 to 9 days. In contrast, spleen cells from C3H/HeN mice that had acquired X5563-specific immunity by intradermal (i.d.) inoculation of viable tumor cells, followed by surgical resection of the tumor, exhibited an appreciable inhibitory effect on the growth of X5563 tumor cells admixed in the chamber. This antitumor effect was mediated by Lyt-1+2-T cells and was tumor-specific, because the growth of X5563 or another syngeneic MH134 hepatoma cells was inhibited by spleen cells from C3H/HeN mice immunized to the respective tumor cell types. Most important, these tumor-specific Lyt-1+2-T cells lost their antitumor activity by depleting an adherent cell population contained in spleen cells, indicating that adherent cells are required for the Lyt-1+2-T cell-mediated antitumor effect. This was substantiated by the fact that immune spleen cells depleted of adherent cells could regain their tumor-inhibiting effect when normal spleen cells were added back as an adherent cell source, or more directly by adding back a splenic or peritoneal resident adherent cell population. These results indicate that tumor-specific Lyt-1+2-T cells mediate the tumor growth inhibition and that their antitumor effect depends on the coexistence of an adherent cell population.     

Effect of tumor immunity on the distribution of labeled lymphoid cells in tumor-challenged mice

The distribution of ⁵¹Cr-labeled lymphoid cells from normal mice and mice immunized against a tumor were compared after intravenous inoculation of the labeled cells into normal syngeneic recipients. Spleen cell preparations from immune donors contained increased percentages of spleen and bone marrow-seeking cells, thus suggesting expansion of these cell populations when immunity to a tumor exists. Homing of labeled normal cells in tumor cell-injected normal animals was somewhat different from that seen in tumor cell-inoculated mice that were immunized against the tumor. In the latter case, accumulations of lymph node and spleen cells in recipient lymph nodes and bone marrow were consistently lower. In contrast, lymphoid cells from animals immunized against the tumor were found to accumulate in virtually the same percentages in lymphoid organs of normal and immune recipients. The behavior of lymphoid cell populations from thymus or bone marrow that consist mainly of precursor cells was unaffected by presence of malignancy and/or tumor immunity.     

Host immune responses to tumor cells augmented by bleomycin and their therapeutic effects on rat fibrosarcoma

The administration--timing-dependent therapeutic effects of bleomycin (BLM) were observed on a fibrosarcoma implanted SC in WKA rats. Five consecutive IP administrations of BLM (5 mg/kg/d) were found to be more effective when BLM was given from Day 8 than when it was given from Day 1 for tumors implanted on Day 0. The therapeutic effects correlated well with antitumor immune responses, which were examined on Day 13 when the tumor had not yet regressed even in surviving rats. The tumor-neutralizing activity of spleen cells was augmented in rats treated with BLM from Day 8 to Day 12, and the suppressor cell activity detected in the spleen cells of tumor-bearing rats was eliminated by the BLM treatment. The tumoricidal activity of peritoneal exudate cells (PEC) was detected in rats treated from Day 8 but not in rats untreated or treated from Day 1. The in vitro treatment of KMT-17 cells with BLM (20 micrograms/ml) for two hours enhanced the sensitivity of the tumor cells to the activity of tumoricidal PEC. This suggests that the direct action of BLM on tumor cells also plays an immunologic role in BLM treatment. The findings reveal that the therapeutic effect of BLM is elicited by its ability to augment the host immune responses to tumor cells.     

Purified mouse mammary tumor and lymphoid cells in immune assays

Summary Tumor and lymphoid cell components from primary mammary adenocarcinomas of C3H/He mice were isolated simultaneously by velocity gradients. Viable tumor cells were obtained in sufficient numbers to test their in vivo and in vitro growth. Isolated tumor cells grew in 97% of inoculated syngeneic animals. In six assays with different tumors the effects of tumor-associated lymphoid cells (TAL) on in vivo tumor growth varied, enhancing in three and delaying in two experiments. Isolated tumor cells from animals with enhancing TAL grew faster in nonirradiated mice, whereas tumor cells from animals with inhibitory TAL grew better in irradiated animals. Isolated tumor cells also proliferated in cell culture, where they averaged 35% primary plating efficiency. Separated tumor cells were used in short-term ⁵¹Cr-release assays with TAL, tumor-bearer lymph node and spleen effectors. Cytotoxicity was detected in only five of 25 assays. In no case was there killing by lymphocyte populations from normal animals. In the present report we describe a technique for the isolation of viable tumor and lymphoid cells from murine adenocarcinomas that allows study of interactions between these populations from the original tumor-bearing host.Postdoctoral fellow of the Fogarty International Foundation. Present address: Department of Surgery, Harvard Medical School and Brigham & Women's Hospital, Boston, MA 02215, USA     

Functional analysis of mononuclear cells infiltrating into tumors. II. Differential ability of mononuclear cells obtained from various tissues to produce helper factors that are involved in the generation of cytotoxic cells

The requirement of CGF in the generation of cytotoxic cells against syngeneic tumor cells (T-9) and in the rejection of transplanted T-9 cells has been investigated. Spleen cells obtained from sensitized rats showed strong cytotoxicity against 51Cr-labeled T-9 cells upon incubation with CGF for 48 hr. Human recombinant IL 2 and rat IFN failed to generate cytotoxic cells from spleen cells of sensitized rats. CGF are produced by spleen cells upon inoculation of T-9 cells into sensitized rats as a host in vivo immune response. Production of CGF preceded the appearance of cytotoxic cells in regional lymph node and tumor tissues. In those rats, inoculated tumor cells were eventually rejected. In contrast, spleen cells failed to produce CGF upon inoculation of T-9 cells in unsensitized rats. Cytotoxic cells were not detected in unsensitized rats, and inoculated tumor grew in those rats. Thus, CGF is likely to be involved in the generation of cytotoxic cells and in the rejection of inoculated syngeneic tumor cells. A Mono Q anion-exchange column with an FPLC system allowed the chromatographic separation of CGF from IL 1, IL 2, IL 3, and CSF.     

The antimetastatic function of concomitant antitumor immunity. I. Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay

A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2b X H-2d) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2d) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2b serum and complement, in order to selectively destroy H-2bd host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2d P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant.     

In situ lymphoid cells of mouse mammary tumors. IV. Comparison of functional activity of lymphoid cells separated from mammary tumors to that of spleen and lymph node cells of tumor-sensitized mice.

Lymphoid cells isolated form several types of mouse mammary tumors are capable of stimulating tumor cell growth or survival in MCT assays. Lymph node and spleen cells of mice bearing such a tumor are specifically cytotoxic to the tumor cells. Surgical removal of the tumor is followed in 4 to 7 days by the appearance of stimulatory capacity in spleens and lymph nodes. By day 10, cytotoxic cells specific for the sensitizing tumor are again detected. These reach a peak on day 13. By day 17 no reactivity is detectable. The functional distribution of tumor-reactive lymphoid cells is different between tumor masses and peripheral lymphoid organs.     

Characteristics of the immunosuppressive activity of lymphoid organ cells in the process of sensitization with ram erythrocytes and exposure to antilymphocyte serum

In the adoptive transfer of cells obtained from the thymus, lymph nodes and the spleen to intact syngeneic animals the suppression of immune response was induced by lymph node cells. If the donors were previously sensitized, the cells of the thymus and lymph nodes showed suppressive activity in the adoptive transfer test. A single injection of antilymphocytic serum to the donors of lymphoid cells, previously sensitized with sheep red blood cells, enhanced the immunosuppressing action of thymocytes and lymph node cells.     

Development of highly immunogenic variants of a rat fibrosarcoma line during in vitro cultivation

Summary Rat firosarcoma KMT-17 cells descreased in tumorigenicity when cultured in vitro. Eight clones derived from cultured KMT-17 cell lines (c-KMT-17) were examined for their tumorigenicity, immunosensitivity, and immunogenicity. All the clones were less or nontumorigenic in normal syngeneic rats than KMT-17 cells maintained in vivo. All eight clones produced tumors in rats immuno-suppressed with 600 rad ⁶⁰Co; differences in degree of tumorigenicity were seen among clones in rats irradiated with 250 rad ⁶⁰Co. Although immunosensitivity of the eight clones to complement-dependent and cell-mediated cytotoxicity was the same or less than that of KMT-17 cells, al leight clones induced greater transplantation resistance to KMT-17 than KMT-17 itself. Cold target inhibition tests demonstrated new antigens in a highly immunogenic variant in addition to the original tumor associated antigen (TAA). New glycolipids, not observed in KMT-17 cells, were demonstrated in the clones by thin layer chromatography. These results suggest that new antigens appearing during culture of KMT-17 may act as helper antigens for TAA, increasing the immunogenicity and decreasing the tumorigenicity of the cultured cells.This work was partially supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science und Culture, Japan.     

Cell cooperation in abolition of tolerance of xenogeneic tumor.

Thymectomized, lethally irradiated mice reconstituted with syngeneic bone marrow cells are tolerant to xenogeneic Yoshida ascites sarcoma (YAS). The tolerance was abolished by an injection of syngeneic normal spleen, thymus, or lymph node cells given simultaneously with YAS. Allogeneic and semiallogeneic spleen cells were ineffective. The YAS-rejecting mice produced specific anti-tumor antibodies. The serum of these mice transferred to tolerant T-cell-deficient mice protected the latter from inoculated YAS cells. These serum-protected mice were not able to resist the reinoculum of the tumor cells as the mice restored with lymphoid cells did. The latter mice rejected the YAS at the time when donor cells were practically absent in their lymphoid tissue. The low effective ratio of injected syngeneic lymphoid to tumor cells, efficiency of injected thymus cells, and other data led to the conclusion that transferred lymphoid cells did not act directly on tumor cells but through cooperation with host lymphoid cells. The cooperation of donor T- and host B-lymphocytes enabled the activation of the latter, and YAS cells were rejected.     

Augmentation of immune response to syngeneic fibrosarcoma T241 with in vivo protection

Summary Lewis T241 fibrosarcoma, a syngeneic tumor in C57 BL/6J mice, was found to be poorly immunogenic. When tumor-bearing animals (TBA) were challenged with tumor cells either concomitantly or after excision of a growing tumor no protection was observed. In vivo (Winn) neutralization assays also showed a lack of tumor immunogenicity. However, in vitro studies showed that a significant proliferative response could be elicited from the spleen cells of TBA when these cells were cultured with either mitomycin-C-treated tumor cells or KCl tumor extract. Similarly, macrophage migration inhibition factor (MIF) was produced by TBA spleen cells upon incubation with KCl tumor extract, but no cell-mediated cytotoxicity to T241 target cells was observed with various lymphoid cell populations at any stage of tumor growth. Immunization of syngeneic animals with Vibrio cholerae neuraminidase(VCN)-treated, irradiated tumor cells alone or admixed with Freund's complete adjuvant (FCA) resulted in decreased tumor growth and fewer pulmonary metastases following challenge with 10⁶ tumor cells. No complete tumor rejection was observed. In contrast, 13 of 16 animals immunized with irradiated tumor cells admixed with FCA rejected 10⁵ tumor cells. Animals that grew tumors had significantly reduced tumor growths and pulmonary metastases. Lymph node and peritoneal exudate cells (PEC) of immunized animals showed significant cytotoxicity to T241 cells.     

The use of antifibrin antibodies for the destruction of tumor cells

Summary The transplantable methylcholanthrene-induced sarcoma (MC-D) in strain 13 guinea-pigs was used to test the hypothesis that tumor cells growing within a fibrin matrix could be destroyed by an immunologically specific strategy involving an indirect cell-mediated immune reaction. The experimental design consisted of two steps: (1) in vivo fixation of anti-guinea pig fibrin antibodies (AGFA) on the fibrin matrix enmeshing the tumor cells, and (2) the reaction between AGFA fixed to the fibrin matrix and lymphoid cells from syngeneic animals sensitized to xenogeneic immunoglobulins isotypic with AGFA. Indeed, tests with ⁵¹Cr-labelled lymphoid cells yielded evidence for the localization of these sensitized lymphoid cells within the fibrin lattice when the latter was coated with AGFA. Moreover, significant tumor growth suppression (P<0.01) was achieved in guinea-pigs that had received rabbit or goat AGFA intravenously and lymphoid cells from syngeneic guinea-pigs sensitized to a state of cell-mediated immunity to rabbit or goat IgG by the subcutaneous route. On the other hand, the administration of the antibodies or of the sensitized cells alone did not affect the growth of the tumor. Preliminary results suggest that peritoneal exudate cells may have an important role in the success of this strategy for tumor cell destruction.     

Growth-inhibitory activity of lymphoid cell plasma membranes. I. Inhibition of lymphocyte and lymphoid tumor cell growth

Membranes isolated from normal spleen cells or lymphoid tumor cells were found to inhibit in vitro growth of several murine tumor cell lines including a B cell hybridoma, a thymoma, and a mastocytoma. 50% inhibition occurred at membrane protein concentrations of 60-100 micrograms/ml. A similar concentration dependence was found for inhibition of [3H]-thymidine incorporation by tumor cells and for the lipopolysaccharide-induced mitogenic response of normal spleen cells. The inhibitory activity co-purified with the plasma membrane upon fractionation of crude membranes. Membrane solubilization with deoxycholate followed by dialysis to remove the detergent gave good recovery of inhibitory activity in the resulting reconstituted membranes. Membrane-mediated growth inhibition resulted from a decreased rate of proliferation and not from increased cell death. A toxic effect of the membranes was further ruled out by the finding that increasing the fetal calf serum content of the medium could substantially reverse the growth inhibition. Thus, the plasma membrane of lymphoid cells contains a component that can slow or stop the growth of cells in culture. This membrane component may have a role in cell contact-mediated regulation of growth.     

The antimetastatic function of concomitant antitumor immunity. II. Evidence that the generation of Ly-1+2+ effector T cells temporarily causes the destruction of already disseminated tumor cells

Progressive growth of the P815 mastocytoma in an immunocompetent host evokes the generation of an antitumor immune response that can be measured in terms of the production of cytolytic Ly-1+2+ T cells in the draining lymph node and spleen. This immunity, designated concomitant immunity, is present on day 6 of tumor growth, peaks on day 9, and decays progressively thereafter. It fails to develop in mice made T cell deficient by thymectomy and lethal whole-body gamma-radiation, and reconstituted with syngeneic bone marrow cells (TXB mice). Employment of a mouse survival assay, capable of enumerating metastatic P815 cells in cell suspensions, showed that the P815 tumor metastasizes to the draining lymph node and spleen at the same rate in normal and TXB mice for the first 6 days of growth of an intradermal P815 tumor. By day 6 of tumor growth there were approximately 10(3) P815 cells in the draining lymph node in both types of mice. However, during the generation of concomitant immunity between days 6 and 9, the number of metastatic P815 cells in the draining lymph nodes and spleens of normal tumor-bearing mice declined by nearly 90%. After day 12, however, the number of tumor cells in the nodes and spleens increased concordantly with the decay of concomitant immunity. These findings, together with the demonstration that T cell-deficient mice failed to restrain the number of metastatic P815 cells in the draining lymph node and spleen, suggest that concomitant immunity is an important defense mechanism against the development of systemic disease. Additional evidence consistent with this interpretation was provided by studies which showed that adoptive immunization with spleen cells from concomitant immune donors significantly prolonged the median survival time of TXB tumor-bearing mice by destroying a substantial proportion of P815 tumor cells already seeded in the draining lymph node. Adoptive immunization also delayed the appearance of metastatic tumor cells in the spleen.     

Specific T-cell factor production and lymphocytes in the direct surroundings of a subcutaneous allogeneic tumor.

Antigen-specific T-cell factors (TCF) play a role in the initiation of cellular immune responses. In allogeneic mouse-tumor models lymphocytes from the direct tumor surroundings of both euthymic and nude mice produce TCF. These lymphocytes produce TCF when collected already 1 day after subcutaneous (sc) injection of tumor cells. In contrast to euthymic mice, draining lymph nodes and spleen of nude mice did not contain TCF-producing lymphocytes at any stage after sc tumor cell injection. In sensitized euthymic mice TCF production by lymphocytes is significantly higher in the direct tumor surroundings than in draining lymph nodes or spleen. At 2 and 5 days after tumor cell injection, the mononuclear cell infiltrate of the tissue surrounding the tumor in euthymic mice showed low expression of Thy 1, CD3, TCR alpha beta, TCR gamma delta, CD4, CD8, and asialo GM1, whereas several lymphocytes and mast cells were positive for monoclonal antibody (mAb) 14-30 (directed against TCF). In both euthymic and nude mice, sc injected tumor cells showed apoptosis. In conclusion, the direct tumor surroundings are the first (and, for nude mice, the only) site of TCF production, sc injection of tumor cells attracts mAb 14-30-positive lymphocytes and renders mast cells positive for mAb 14-30.