The lymphocyte response to primary Moloney sarcoma virus tumors: definition of a non-specific component of the in vitro cellular hyporeactivity of tumor-bearing hosts

Adult CBA mice injected with Moloney Sarcoma Virus (MSV) developed tumors within 5 to 8 days which usually completely regressed within 1 month. Maximal tumor size was variable, occurring between 10 and 15 days. Lymphocytes from these animals were examined for activity against syngeneic target cells bearing the Moloney Leukemia Virus (MLV) -associate dcell-surface antigen. As previously found with BALB/c mice, the lymphocytes were found to be active in microcytotoxicity tests against these target cells; however, the animals which were bearing tumors at the time of testing were found to have the least active lymphocytes. These animals were also immunized against H-2^d antigens 10 to 13 days prior to testing and their lymphocytes tested for cytotoxicity against H-2^d target cells in ⁵¹Cr release assays. It was found that the tumor-bearing animals, by day 10-16 after MSV infection, had lymphocytes which were significantly less active against the H-2^d antigens than those of animals tested prior to the development of tumor or those in which the tumors had regressed. It was thus concluded that a component of the cellular hyporeactivity, as measured by in vitro cytotoxicity in these primary MSV tumor bearing hosts, is non-specific.     

The immune response to primary Moloney sarcoma virus tumors in BALB-c mice: cellular and humoral activity of long-term regressors

Adult BALB/c mice injected with Moloney sarcoma virus (MSV) developed local tumors at the site of inoculation which spontaneously regressed within 20–25 days after injection. Lymphocytes and sera from long-term regressor animals were examined for their specific activities in vitro against Moloney leukemia virus (MLV) determined antigen (s). Specific activity against the MLV-positive target cells by the lymphocytes from these animals was found to be dependent on the presence of B lymphocytes. In order to investigate some of the possible mechanisms of action of the immune B cells, sera, which were characterized by complement-dependent cytotoxicity, immunofluorescence and virus neutralization, were tested for their ability to stimulate normal syngeneic lymphocytes to be active against the target cells. These antisera placed on the target cells were found to stimulate normal unfractionated or T deprived cells to reduce the target-cell numbers. This effect was not found with normal T lymphocytes. Time-course kinetics of this antibody-dependent cell-mediated activity in vitro were defined.     

The humoral antibody response to a primary viral neoplasm (MSV) through its entire course in BALB-c mice

BALB/c mice injected with Moloney sarcoma virus developed within 5–12 days tumors at the site of inoculation which usually completely regressed by day 20–25. Maximal tumor size was reached around day 15. Sera from these animals were examined for complement-dependent cytotoxic activity against Moloney lymphoma cells. The cytotoxic antibody response was biphasic. The first peak was reached at day 10 and extended to day 15 (peak tumor size). Twenty days after virus infection in the early regressor sera, antibody titres fell to normal serum values, followed by a progressive rise which persisted in the long-term regressors during the entire observation period of 3 to 6 months. Active sera from days 10, 15, 30 and long-term regressors were pooled separately and fractionated on Sephadex G-200 into 19S and 7S fractions, and reconcentrated to the original volume. The fractions were then tested for cytotoxic activity. Both 19S and 7S fractions of all pools contained cytotoxic activity. Immunofluorescence analysis of these fractions showed the 7S fractions to be detectable by membrane immunofluorescence but not the 19S. Virus neutralizing activity was found in both 19S and 7S fractions with higher activity in the sera from regressor animals.     

Effect of recombinant human interleukin 2 on the growth of a BALB/c sarcoma induced by Moloney murine sarcoma virus

The effect of in vivo administration of recombinant interleukin 2 (rIL2) on the growth of a primary female BALB/c sarcoma induced by Moloney murine sarcoma virus (M-MSV) was studied. Although low-dose administration of (6,000 JU/mouse x 14 days) rIL2 had no effect on the growth of the tumors, high-dose (15,000-80,000 JU/mouse x 14 days) intraperitoneal inoculation of rIL2 induced tumor regression, dose-dependently. Tumors in mice which received 80,000 JU/mouse/day of rIL2 regressed completely 2 weeks after the initiation of treatment. The survival rates of the treated groups were significantly higher than those of the control group. A time course experiment disclosed that the effect of rIL2 was restricted only to the group in which rIL2 treatment started 8 days after the inoculation of M-MSV. The cytotoxic activity of regional lymph node lymphocytes from rIL2-treated mice was demonstrated against primary culture of M-MSV-induced sarcoma but not against syngeneic tumor induced by methylcholanthrene (Meth A). The effect of rIL2 was partially blocked by the administration of anti-IL2 receptor antibody. Immunohistochemical examination revealed that infiltration of Thy1.2+Lyt1+2- (helper/inducer subset) lymphocytes into the tumor tissue was prominent in mice which received high-dose rIL2. The results indicated that IL2 induced regression of M-MSV-induced sarcoma mainly through activation of IL2-receptor-positive helper T cells in the tumor tissues and of killer cells in the draining lymph nodes.     

Effect of Recombinant Human Interleukin 2 on the Growth of a BALB/c Sarcoma Induced by Moloney Murine Sarcoma Virus

The effect of in vivo administration of recombinant interleukin 2 (rIL2) on the growth of a primary female BALB/c sarcoma induced by Moloney marine sarcoma virus (M-MSV) was studied. Although low-dose administration of (6,000 JU/mouse × 14 days) rIL2 had no effect on the growth of the tumors, high-dose (15,000–80,000 JU/mouse × 14 days) intraperitoneal inoculation of rIL2 induced tumor regression, dose-dependently. Tumors in mice which received 80,000 JU/mouse/day of rIL2 regressed completely 2 weeks after the initiation of treatment. The survival rates of the treated groups were significantly higher than those of the control group. A time course experiment disclosed that the effect of rIL2 was restricted only to the group in which rIL2 treatment started 8 days after the inoculation of M-MSV. The cytotoxic activity of regional lymph node lymphocytes from rIL2-treated mice was demonstrated against primary culture of M-MSV-induced sarcoma but not against syngeneic tumor induced by methylcholanthrene (Meth A). The effect of rIL2 was partially blocked by the administration of anti-IL2 receptor antibody. Immunohistochemical examination revealed that infiltration of Thy1.2⁺Lyt1⁺2^- (helper/inducer subset) lymphocytes into the tumor tissue was prominent in mice which received high-dose rIL2. The results indicated that IL2 induced regression of M-MSV-induced sarcoma mainly through activation of IL2-receptor-positive helper T cells in the tumor tissues and of killer cells in the draining lymph nodes.     

Studies on cellular immunity and its serum mediated inhibition in Moloney-virus-induced mouse sarcomas

A colony-inhibition technique was used to demonstrate lymph-node cell (LNC) mediated immune reactions against tumor-specific transplantation antigens of primary, Moloney sarcoma virus induced mouse sarcomas. Lymph-node cells from mice in which Moloney sarcomas had regressed spontaneously (regressors), as well as LNC from mice carrying progressively growing tumors (progressors), induced by virus inoculation at an age of 14 days or older, reduced the plating efficiency of Moloney sarcoma target cells. Serum from mice with progressively growing Moloney sarcomas, but not from mice with spontaneous mammary carcinomas or methylcholanthrene-induced sarcomas, abrogated the inhibitory effect of regressor LNC on Moloney sarcoma cells. Additional evidence for the specificity of the serum effect was obtained in experiments showing that the protective effect of progressor sera could be specifically removed by absorption with Moloney sarcoma cells. Regressor sera gave no protection against target cell inhibition by regressor LNC. It is suggested that progressor sera contain antibodies which mediate an efferent form of immunological enhancement.     

Antibody-dependent, cell-mediated cytotoxicity with autochthonous lymphocytes and sera after infection with Moloney sarcoma virus.

Antibody-dependent, cell-mediated cytotoxicity in the Moloney sarcoma virus (MSV) system was analyzed in terms of the ability of autochthonous antibody to induce or potentiate cytotoxicity by lymphocytes from animals infected with MSV. As previously demonstrated in microcytotoxicity assays, the lymphocytes from regressor animals taken 30 days after virus infection were consistently more cytotoxic than those from tumor-bearing animals 15 days after infection. Antisera from the regressors potentiated the activity of regressor lymphocytes from the same animals. Also, antisera from tumor bearers, 15 days after virus injection, induced cytotoxicity by the animals' autochthonous lymphocytes which, by themselves, were not cytotoxic. In an independent assay for antibody, both groups of sera produced cytotoxicity by control nonimmune lymphocytes. Specificity controls indicated that both antibody and lymphocytes were required for the induction of cytotoxicity against the target cells in vitro. Normal sera placed on the target cells in the same concentrations induced no cytotoxicity by the immune lymphocytes, and immune sera alone placed on the target cells caused no cytotoxicity. The cooperative activity between antibody and lymphocytes may be a factor that accounts for the observed high incidence of spontaneous tumor regression.     

Cell-mediated reactivity to antigens shared by Moloney-virus-induced lymphomas (LSTRA) and certain 3-methylcholanthrene-induced mouse sarcomas.

Spleen cells (SC) both from BALB/c mice whose primary Moloney sarcoma virus (MSV)-induced sarcomas had spontaneously regressed and from normal, untreated BALB/c mice, were co-cultivated for 5 days with mitomycin-C-treated LSTRA cells; LSTRA is a BALB/c Moloney lymphoma which shares cell surface antigens with MSV-indiced sarcomas. These SC, referred to as CMR and CU cells, respectively, were shown to be cytotoxic to LSTRA cells in 3 h 51Cr-release assays; CMR cells showed, in most cases, the greatest lytic activity against LSTRA targets. The same SC were also reactive, in 20-h microcytotoxicity and 51Crassays, against target cells from a variety of transplanted sarcomas indiced by 3-methylcholanthrene (MCA) in Balb/c mice. The highest reactivity was seen when CMR or CU cells were tested against target cells from sarcoma lines that expressed an NB-ecotropic MuLV cross-reacting serologically with Moloney virus. Reactivity against isotope-labelled tumor cells expressing MuLV-associated cell surface antigens could be competititively inhibited by adding unlabelled tumor cells expressing such antigens. Finally, Winn assays were performed in which CMR cells strongly inhibited the outgrowth of cells from three sarcoma lines that express the NB-ecotropic MuLV. There was less but significant inhibition of cells from some other MCA sarcomas, either negative for the expression of MuLV-associated antigens or expressing the N-ecotropic endogenous BALB/c MuLV. CU cells enhanced tumor outgrowth in Winn assays at least as often as they inhibited it.     

Stimulation of tumor growth by the immune response

An attempt has been made to test the immunostimulation hypothesis of Prehn which proposes that, in its initial stages, the immune response to tumour-specific antigens stimulates rather than inhibits tumor growth. Mice have been injected with cells from three different methylcholanthrene-induced tumors and the cellular and humoral anti-tumor immune responses of their peripheral blood lymphocytes and sera have been studied by in vitro techniques. The results have been found to be completely compatible with the immunostimulation hypothesis. Five days after the inoculation of tumor cells, at a time when tumors are still not palpable, the cellular anti-tumor immune responses of the mice were found to be capable of specifically stimulating tumor growth. Palpable tumors normally appeared in these animals 7–10 days after the inoculation of tumor cells and by day 12 it was found that, in most instances, the stimulatory pattern seen on day 5 had changed to an inhibitory one. Other findings reported in this paper relate to the effect of normal and immune serum on tumor cells and on the interaction between tumor cells and normal or immune lymphocytes. Finally, on the basis of all these results, a hypothesis is presented to explain how tumors grow and why they do so in the face of an immune response directed against antigens specific to them.     

Immune reactivity in the Moloney strain of murine sarcoma virus oncogenesis: requirement of thymus-derived lymphocytes for in vivo protection.

To study the function of different lymphocyte populations in the Moloney strain of murine sarcoma virus (M-MuSV) tumorigenesis, we gave M-MuSV injections to CBA mice selectively deprived of thymus (T) lymphocytes by thymectomy, X-rradiation, and syngeneic bone marrow injection. Although no tumors appeared in the control group, 80% of the derived mice had tumors that grew progressively and ultimately killed them. In deprived mice, grafted with a syngeneic thymus (reconstituted mice) before or after an M-MuSV injection, tumors regressed or did not develop. Histologically, the lymph nodes and spleens of reconstituted mice, compared to those of deprived animals, showed repopulation of the thymus-dependent areas and prominent follicles in the cortex. Moreover, tumor tissue of reconstituted mice was extensively infiltrated by lymphocytes. To evaluate the number of lymphoid cells needed to prevent or regress M-MuSV tumors, we injected varying amounts of lymphoid cells into deprived mice. Even low lymphocyte numbers (10(6) cells) were sufficient to exert, in some cases, protection against M-MuSV tumorigenesis. This effect was not abolished by subsequent splenectomy or antilymphocyte serum treatment. Finally, deprived mice, given repeated injections of antiserum (hyperimmune) against M-MuSV, had tumors which appeared only after a prolonged latency. From these results, it is concluded that T-cell population integrity is important in affording total host protection against the M-MuSV tumors.     

Adoptive chemoimmunotherapy of a moloney lymphoma

Adult BALB/c mice were inoculated with BALB/c Moloney lymphoma cells. On day 6, mice with palpable tumors received sublethal cyclophosphamide (CY) and BALB/c or DBA 2 spleen cells. All untreated mice died with tumor on days 8-12. Spleen cells alone had no effect. All mice treated with CY alone died with tumor by day 30, as did all mice treated with CY plus cells from non-immune donors or from DBA donors immunized against normal BALB/c tissue. By contrast, treatment with CY plus cells from BALB/c or DBA/2 donors immunized with antigenically-related murine sarcoma virus (Moloney) was moderately effective — 30 of 65 mice lived beyond day 60 and 14 of 65 beyond day 100. Lethal X-irradiation of the immune cells abolished their efficacy. Thus, an established antigenic lymphoma was inhibited or eradicated by a combination of chemotherapy and immunotherapy with spleen cells from syngeneic or H-2-compatible donors, but only if the cells were viable and were derived from donors pre-immunized against tumor-associated antigens. Finally, when lethal X-irradiation was substituted for sublethal CY, immunotherapy was not effective, probably because the tumor cells were far less sensitive to X-ray than to CY.     

Anti-tumor immunity in b-lymphocyte-deprived mice. iii. immunity to primary moloney sarcoma virus-induced tumors

Tumor induction and immunity to tumors were studied following the injection of Moloney sarcoma virus (MSV) into mice whose B-lymphocyte functions had been suppressed by the chronic administration of anti-IgM antibodies. Two preparations of MSV were used; one which gives rise to tumors which uniformly regress in normal adult mice, and another which elicits progressively growing tumors in the majority of recipients. The tumor incidence, mean tumor size, and tempo of regression were not modified by treatment with anti-IgM. However, whereas tumors induced by the regressor virus were all rejected in 19 NRG-treated and 29 untreated recipients, continued growth was obtained in 2 of 23 B-lymphocytedeprived mice. Furthermore, in 9 additional mice from this group, apparent rejection was followed by tumor recurrence at the site of the initial tumor. Continued growth was accompanied by widespread metastasis. These tumors were freely transplantable to normal syngeneic recipients. Metastasis and transplantability were also detected in 7 of 24 anti-IgM-treated mice given progressor virus, but were not seen in the control animals. Recurrence and metastasis was obtained despite the presence of high levels of specific cytotoxic T lymphocytes in the spleen. It is concluded that B lymphocytes or their products play an essential role in host protection against MSV-induced tumors.     

Suppression of Moloney sarcoma virus immunity following sensitization with attenuated virus.

Murine sarcoma virus (Moloney strain) (MSV-M)-induced tumors are unusual in that they regularly appear less than 2 weeks after virus inoculation, progress for 1 to 2 weeks, and are rejected by normal adult BALB/c mice. Rejectio leaves the animals immune to tumor induction. In the present study, presensitization of normal adult BALB/c mice with attenuated MSV-M resulted in an altered pattern of tumor immunity. Injection of active MSV-M into the presensitized animals resulted in tumor induction and rejection similar to that observed in normal animals, but rejection failed to produce protection against the secondary inoculation with MSV-M. After the second inoculation with active MSV-M, tumors appeared and progressed but ultimately were rejected. Over 80% of the mice died, 25% after the primary challenge and the remainder after the secondary challenge. At death, all mice had histological evidence of leukemia which was the probable cause of death. The animals that died following the secondary challenge also had evidence of disseminated MSV-M. Solid tumor nodules were found in skeletal muscle distant from the original site of inoculation, and active MSV-M was isolated from spleen and lungs. The possibility that the results were produced by specific suppression of MSV-Moloney leukemia virus immunity is discussed.     

The cellular events associated with regression and progression of murine (Moloney) sarcomas

Tumors were induced in adult and neonatal mice by intramuscular injections of either 10⁴ or 10⁶ cells from a cultured murine (Moloney) sarcoma line. Neoplasms that progressed were induced in neonates by either dose, and in adults only by the larger dose; adult mice receiving 10⁴ cells usually developed tumors that regressed. Light microscopic examinations were performed at 2–3 day intervals throughout the course of tumor development and subsequent regression or progression. Initially all tumors became infiltrated with polymorphonuclear leukocytes—mainly neutrophils—and edema was extensive. By the end of the second week post inoculation, this acute inflammatory infiltrate had been replaced in adult mice by one consisting of mononuclear cells; neonatal mice never developed significant numbers of these inflammatory cells in their tumors. Of particular significance, since mononuclear inflammatory cells were associated intimately with tumors during the process of regression, was the disappearance of these cells 12–14 days post inoculation from tumors destined to progress in adult mice. Hyperplastic changes were found in the cortices and medullae of regional lymph nodes draining both progressing and regressing sarcomas. Secondary neoplasms developed commonly, and the distribution of these lesions was related to the ages of mice at the time of inoculation.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. V. Transfer of protective immunity with H-2 identical immune T cells from B10.D2 mice.

Evidence is presented for the existence of immune T lymphocytes which have protective activity in vivo against the lethal effects of a metastasizing tumor. ESb, the metastasizing tumor cell line of our model system, which is highly malignant in syngeneic DBA/2 mice, can be rejected when transplanted into B10.D2 mice. Since B10.D2 and DBA/2 mice are identical at the H-2 complex, cells could be transferred from the tumor-resistant into the susceptible strain to determine whether they could confer protective immunity in a syngeneic environment containing disseminated ESb tumor cells. Protective immunity against ESb could be transferred into DBA/2 with spleen cells but not with ascites fluid from B10.D2 mice preimmunized against the ESb tumor cells. B10.D2 spleen cells taken between 6 and 22 days after tumor transplantation had protective immunity, while cells taken from animals 3 days after tumor transplantation, or from normal animals, had no significant protective effect. The cells in B10.D2 immune spleens with protective activity were sensitive to treatment with anti-theta serum and complement and did not adhere to nylon-wool columns. Such nylon-wool column-passed cells which were enriched for T lymphocytes had a higher protective activity then the unfractionated cells. DBA/2 mice which received viable ESb tumor cells subcutaneously on day 0 could be protected by intravenous inoculation of B10.D2 immune spleen cells only when these were given after the tumor cells (day +1, +3, +5, +7), not when given before (day ?3 or ?1). The mechanisms of protective immunity against tumor metastasis and the significance of our findings for completely syngeneic tumor systems will be discussed.     

Effects of levamisole on in vivo and in vitro murine host response to syngeneic transplantable tumor.

The effects of levamisole were studied in vivo and in vitro on two murine tumors, B16 melanoma and adenocarcinoma 15091, syngeneic to the mouse strains used. Administration of levamisole before tumor transplantation enhanced the early appearance of neoplasms but did not affect the overall incidence or course of tumor growth as compared with that observed in controls given saline injections or animals given levamisole with lethally X-irradiated tumor cells. Administration of the drug 1 day before iv injection of tumor cells significantly reduced the incidence of pulmonary nodules, but if the drug was given 3 or 5 days before tumor challenge, the incidence of nodules was increased. Lymphocytes or macrophages from normal mice given levamisole had no effect on tumor cells in vitro, whereas lymphocytes incubated with levamisole in vitro enhanced tumor cell growth. When lymphocytes and tumor cells were mixed in vitro, lymphocytes from animals treated with the drug formed larger multicell clumps with tumor cells than did those from normal controls. We concluded that levamisole did not protect the mice against the tested tumors.     

Systemic adoptive transfer of immunity and low-dose irradiation eradicate metastases of 13762A rat mammary adenocarcinoma

Rats cured of poorly immunogenic 13762A tumor by a combination of surgery and cyclophosphamide (CY) treatments produced peritoneal exudate cells (PEC) that prevented tumor growth when transferred to naive recipients, but they were ineffective against established tumor. A highly immunogenic 13762A clone (18A) induced PEC lymphocytes that completely reversed the growth of established primary tumor and of lymph-node metastases. 18A-immune PEC alone strongly inhibited tumors of 7 days' duration, but only moderately suppressed 14-day tumors, and had no effect on 21-day tumors. Irradiation (450 R) of rats prior to tumor transplantation improved the effectiveness of the PEC given at 7 days, but the benefit had gone by 14 days. Long-term T-cell depletion prior to tumor challenge allowed PEC inhibition of 7- and 14-day tumors, but not 21-day tumors. The most potent strategy was the administration of 450 R followed immediately by immune PEC. When rats with 21-day tumors were so treated, the metastases grew temporarily to a maximum diameter of 2-5 cm and then completely regressed. We concluded that a combination of immune T cells and 450 R can cure established, massive metastases, probably through a combination of an increase in the numbers of T-cell effectors and elimination of suppressor cells.     

Secondary in vitro generation of cytolytic T-lymphocytes (CTL's) in the murine sarcoma virus system. Virus-specific CTL induction across the H-2 barrier.

Following in vitro stimulation of murine sarcoma virus Moloney isolate (M-MuSV)-immune spleen cells with syngeneic antigenically related Moloney leukemia cells, highly efficient cytotoxic T-lymphocytes (CTL's) were generated. The cytotoxic effect was directed only against H-2-compatible target cells bearing M-MuSV tumor-associated antigens (TAA). However, in a cold target competition assay a weak but detectable capacity to block CTL activity was also obtained when allogeneic Moloney leukemia cells were added. Moreover, when M-MuSV-immune spleen cells from mice inoculated with virus 14 days previously were stimulated by allogeneic Moloney leukemia cells, a strong cytotoxic effect toward syngeneic and allogeneic tumor cells bearing M-MuSV TAA was elicited.     

A laboratory model for the study of the immunobiology of osteosarcoma.

Inoculation of Moloney sarcoma virus into the marrow cavity of the tibia of newborn Wistar-Lewis rats resulted in the appearance of an initially localized osteosarcoma in 97.7% of these animals. At least 77.9% of the rats developed lung metastases and died, usually within 6 weeks of inoculation. The remaining 22.1% showed regression of disease after initial growth of the tumor. Tumor cells were maintained in tissue culture and used as target cells for a visual and isotopic (3H-thymidine or 125IUdR) microcytotoxicity assay. Cell-mediated immunity could be measured by these methods throughout the course of the illness in animals with progressive disease as well as in those whose tumors eventually regressed. The presence of serum factors capable of modifying the level of CMI was documented. This Moloney-sarcoma-virus-induced rat osteosarcoma and human osteosarcoma thus appear to have several basic pathologic and immunologic similarities. The model may be useful for studying the effects of a variety of treatment protocols upon the clinical course and immune response to osteosarcoma.     

Role of regional and distant lymph nodes in rejection of feline sarcoma virus-induced tumors in sheep.

The immune response of regional and distant lymph nodes was compared relative to rejection of feline sarcoma virus (FeSV)-induced tumors in sheep. Following injection of FeSV-transformed allogeneic or autochthonous fibroblasts into the lower leg, small tumors developed at the site of inoculation and subsequently regressed. Efferent lymph from the regional popliteal nodes and distant nodes in the same host was collected for periods up to 40 days after tumor cell inoculation. The cell response in the efferent lymph of the stimulated node was the same regardless of whether inoculum consisted of autochthonous or allogeneic FeSV-transformed sheep cells. There was a rapid rise in total lymphocytes leaving the regional node, beginning at 3 days and peaking at 6--8 days post inoculation. On days 6--8 post inoculation, lymphoblasts appeared in regional lymph ranging from 25 to 40% of the total cell output. The cell population in lymph from distant (nonstimulated) nodes, however, remained morphologically normal throughout the response. Lymphocytes cytotoxic to the injected FeSV-transformed cells appeared in efferent lymph from the regional node within 5 days post inoculation and in lymph from distant nonstimulated nodes several days later. Cytotoxic lymphocytic cells had no "killing" effect against the corresponding nontransformed cells if the inoculum was autochthonous in origin; however, they did have such an effect when corresponding nontransformed cells were allogeneic. The cytotoxicity of lymph cells varied according to the type of cells in the lymph. With the use of the growth inhibition assay, it was possible to demonstrate that lymph cell populations high in lymphoblasts "killed" all target cells in 24 hours, whereas populations of lymph cells comprised mainly of small lymphocytes took up to 2--3 days to "kill" the target cells. Complement-dependent antibody first appeared in lymph from the stimulated popliteal node at 8 days post inoculation and at 12 days post inoculation in blood sera and lymph from distant nodes.