Sensitization of T lymphocytes in vitro by syngeneic macrophages fed with tumor antigens.

We investigated the interaction between T lymphocytes and macrophages in the vitro sensitization of lymphocytes against tumor cells. Spleen cells were sensitized in vitro by syngeneic peritoneal macrophages that had been fed with cell-free antigen preparation of syngeneic tumor cells. The sensitized T lymphocytes acquired specific cytotoxic cells. The sensitized T lymphocytes acquired specific cytotoxic activity in vitro and the capacity to inhingeneic fibroblasts, or the antigen preparation by itself were not able to sensitize the lymphocytes against the tumor.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Generation of suppressor lymphocytes during sensitization in culture against a syngeneic tumor: affinity chromatography on insolubilized histamine

We investigated the effect of depletion of histamine-binding lymphoid cells on immunological properties of lymphocytes sensitized in culture against tumor cells. C57BL/6 spleen cells that were sensitized in vitro on monolayers of the syngeneic Lewis lung carcinoma (3LL) became cytotoxic to the tumor cells in vitro after 3 to 5 days of sensitization. Sensitized cells harvested after 4 days of sensitization occasionally enhanced tumor growth in vivo. Fractionation of the sensitized lymphocytes over insolubilized histamine-rabbit serum albumin-Sepharose (HRS) columns decreased or abolished the enhancing activity in vivo and specifically increased the in vitro cytotoxic activity of the depleted lymphocytes. A similar increase in the cytotoxic activity of HRS-fractionated cells was observed in an allogeneic combination of C57BL spleen cells sensitized against C3H fibroblasts. The effect of HRS chromatography on the in vitro cytotoxic activity increased with prolonged incubation of the depleted effector cells with the target cells.     

Recruitment of effector lymphocytes by initiator lymphocytes. Circulating lymphocytes are trapped in the reacting lymph node.

In previous studies, we have shown that initiator T lymphocytes (ITL) sensitized in vitro recruit effector T lymphocytes in vivo. When ITL were sensitized against fibroblast antigens in vitro and injected into footpads of syngeneic recipients, they induced enlargement of the draining popliteal lymph node (PLN) and the development there of specific effector lymphocytes of recipient origin. To study the basis of this lymph node response in recruitment, we injected 51Cr-labeled spleen cells i.v. into recipients of sensitized ITL and found that the labeled circulating lymphocytes were trapped in the reacting PLN. The trapping depended on surface properties of the labeled circulating lymphocytes, as revealed by various enzymatic treatments. The trapping process was radiosensitive, both on the part of the trapped lymphocytes and the lymph node-trapping mechanism. Thus, sensitized ITL injected into the hind footpads migrate to the PLN and induce the trapping of circulating recruitable lymphocytes, which either differentiate into or regulate the differentiation of effector T lymphocytes.     

The immunological basis of tumor rejection: the absolute dependence of the effector arm on sensitized T cells after chemoimmunotherapy of a murine sarcoma

The mechanisms of tumor rejection were investigated by using a therapeutic model system involving treatment of C57BL/6J (B6) mice bearing the syngeneic MCA/76-9 or the unrelated MCA/76-64 sarcomas with cytoxan and tumor-sensitized T lymphocytes. Separated tumor-associated T lymphocytes (TAL) and tumor-associated macrophages (TAM) isolated from the regressing tumors 8 to 10 days after combination therapy expressed relatively specific cytotoxicity in vitro, whereas the unseparated tumor-associated cells (TAC), consisting of a mixture of TAL and TAM, expressed nonspecific cytotoxicity. TAM-mediated cytotoxicity was not dependent on the presence of TAL, as shown by T cell depletion of TAM or TAC cultures with the use of monoclonal anti-Thy-1 or anti-Lyt-2 antibody and complement. In contrast, the nonspecific cytotoxicity was dependent on the presence of T cells. In vivo assays using the Winn test failed to confirm certain aspects of the in vitro data. Without exception, the TAC inhibited tumor growth in an immunologically specific manner, having no effect on the growth of the unrelated B6 sarcoma. T cell depletion completely abrogated in vivo cytotoxicity. Specificity of tumor growth inhibition was confirmed in a bystander experiment in which TAC were mixed with both tumor cell types and were injected into recipient B6 mice. Tumors grew under these conditions, but the tumor that grew consisted only of those tumor cells toward which TAC cytotoxicity was not specifically directed. A bioassay indicated that the specifically immune antitumor effects at the site of regression were initiated between days 3 and 7 after combination therapy. By days 7 and 9, few tumorigenic stem cells could be detected at the tumor site. However, T cell depletion of the TAC isolated on days 8 to 10 resulted in enhanced tumor growth when the depleted TAC were injected into recipient mice. The conclusions reached were that tumor rejection was absolutely dependent on T cell participation at the tumor site, and that if TAM were involved, they required the presence of TAL and did not express nonspecific antitumor cytotoxicity. Indeed, the accelerated tumor growth seen in the absence of TAL suggested the possibility that TAM were growth stimulatory.     

The role of dendritic cells in the initiation of immune responses to contact sensitizers. I. In vivo exposure to antigen

Twenty-four hours after skin painting mice with picryl chloride (PIC) there was a four- to fivefold increase in the numbers of dendritic cells (DC) isolated from the lymph nodes. These DC initiated primary proliferative and cytotoxic responses when added to cultures of normal syngeneic lymph node cells. The proliferative response was enhanced when the donors of the responding lymph node cells were sensitized with the same antigen. Contact sensitivity developed in syngeneic mice injected into the footpads with 30,000-50,000 DC from lymph nodes of mice painted with picryl chloride 1 day previously. Thus, 1 day after skin painting mice, there were dendritic cells in the draining lymph nodes which were able both to initiate primary stimulation of lymphocytes in vitro and to sensitize recipient mice to give specific delayed hypersensitivity reactions.     

Thymus-derived lymphocyte-dependent rejection of syngeneic papovavirus (SV40) and methylcholanthrene tumors in inbred hamsters.

Treatment of specifically sensitized MHA hamster lymphoid cells with rabbit antisera specific for hamster thymus-derived lymphocytes, in the presence of C, eliminated those cells capable of inhibiting the growth of syngeneic SV40 and methylcholanthrene tumors in vivo. Thymectomized, lethally-irradiated, bone marrow-reconstituted hamsters, shown to be devoid to T cell function, were, after attempted specific sensitization to the two syngeneic tumor cell lines, unable to reject either tumor by direct challenge in vivo. In addition, lymphocytes from such animals were incapable of inhibiting the growth of either tumor cell line in normal syngeneic recepients in the tumor cell neutralization assay. These data strongly support the conclusion that specifically sensitized thymus-derived lymphocytes are required for the rejection of syngeneic SV40 and methylcholanthrene tumors in inbred hamsters.     

In vivo natural antitumor resistance against murine EL-4 lymphoma cells in lethally irradiated syngeneic C57Bl/6 mice

Natural resistance has been detected in lethally irradiated C57Bl/6 (B6) mice inoculated intravenously with the ascites form of a syngeneic B6 leukemia. EL-4 cells were injected into lethally irradiated (800 R) B6 mice and tumor cell proliferation was evaluated by 125IUdR uptake in different organs 4 days after the challenge. Differential growth of lymphoma cells was observed when young mice were injected as compared with older mice and when mice were treated with agents known to interfere with natural resistance (e.g., poly(I:C), FLV-P, carrageenan, cyclophosphamide, high doses of irradiated cells). Similar results were obtained by measuring rapid clearance of 125IUdR-labeled EL-4 cells from lungs of intact B6 mice. In vivo cold competition studies, employing EL-4 and several other tumor lines of the same or different haplotype, showed that only EL-4 and RBL-5 cells were capable of inhibiting syngeneic resistance against EL-4 tumor. On the contrary, YAC-1 lymphoma cells, the most susceptible target to natural killer-mediated cytotoxicity in vitro, did not compete. These results suggest that EL-4 cells express membrane determinants not detectable on normal H-2b parental bone marrow cells and are susceptible to natural resistance against hemopoietic tumor cells in lethally irradiated syngeneic B6 mice.     

Morphological and functional characteristics of cells infiltrating and destroying tumor multicellular spheroids in vivo.

EMT6 mammary sarcoma cells were grown in vitro as multicellular spheroids to model for the heterogeneity of microenvironments and structural changes which develop in many tumors, including micrometastases. Spheroids of 700-900 micron diameter were implanted into and recovered at different times from the peritoneal cavities of sensitized or nonsensitized allogeneic and syngeneic mice. The colony forming efficiency of spheroid tumor cells recovered at 24 and 48 h from sensitized allogeneic mice was markedly decreased as compared with those from nonsensitized allogeneic or syngeneic animals. These recovered spheroids were extensively infiltrated by both lymphocytes and macrophages, which ultrastructurally had very close membrane associations with tumor cells. Host cells recovered from spheroids exhibited cytotoxic activity in an in vitro 51Cr release assay. Thus, multicellular spheroids in vivo provide a unique experimental model to study the functional capacity of host cells within a spheroical tumor. Although lacking the stroma and the vasculature of in vivo solid tumors, this model does have many similarities to in vivo tumors and is thus suitable for studying the tumor cell-host cell interactions within the tumor microenvironment. In addition, the system offers the potential for quantitative study of the effects of treatment modalities on tumor cell-host cell interactions.     

Transfer in vivo of cytotoxic immune lymphocytes obtained in vitro in primary culture against a murine lymphoma antigenically altered by DTIC]

Specific cytotoxic T cells were obtained by coculturing "in vitro" normal spleen cells with inactivated histocompatible DTIC-altered lymphoma cells. The "in vivo" antitumor activity of such sensitized lymphocytes was evaluated by injecting a mixture of lymphocytes + tumor into the brains of lethally irradiated syngenic mice. The results indicated that such lymphocytes demonstrate antitumor activity against the same tumor but not against unrelated tumors.     

Immune response to a syngeneic mammary adenocarcinoma. III. Development of memory and suppressor functions modulating cellular cytotoxicity.

Measurement of the development of cytolytic activity by mammary tumor primed or unprimed syngeneic spleen cells on in vitro monolayers of the 13762 rat mammary tumor operationally defined several subpopulations of lymphoid cells involved in the cytotoxic response. In vitro sensitization of cells from Fischer 344 animals injected 2 to 10 days earlier with 2 x 10(7) viable tumor cells always resulted in a higher and earlier lytic response than cells from non-inoculated animals. Adoptive transfer of the same in vivo primed cells for 5 days in irradiated syngeneic hosts removed any cytotoxic cells originally present but subsequent in vitro sensitization still resulted in a higher and earlier cytolytic response. We defined such cells as "memory" cells for cytotoxicity. Memory cells were radiosensitive and specific for the immunizing target cell. In contrast to cells from animals inoculated for 3 to 10 days, cells obtained 11 and 12 days after immunization had a lower response than unprimed cells on vitro sensitization. The anamnestic response could be restored either by culturing 12-day primed cells in vitro for 2 days without antigen or by adoptive transfer for 5 days into irradiated syngeneic rats. This suggests that another population of cells is present in spleen and suppresses the conversion of memory to cytotoxic cells. A more direct measurement of suppressor cell function was obtained by coincubating tumor-primed and unprimed cells on monolayers during in vitro sensitization. Cells from animals bearing tumors for 5 to 10 days always caused an increase in the response of the mixed lymphocyte groups, whereas 11- to 13-day tumor primed cells always caused a marked decrease in the cytolytic response. These results suggest the following interpretation of the kinetics of cell-mediated cytotoxicity to syngeneic tumor inoculation. Cytotoxic cells appear about 6 days after immunization, reach peak levels 2 days later, and then decrease rapidly. Memory cells are generated at a faster rate, reach peak levels before maximum cytolytic activity, but are then functionally inhibited from converting into differentiated cytotoxic cells by a new population of suppressor cells which reach peak activity about 12 days after immunization.     

T-cell-dependent hybrid resistance against a natural killer-resistant Moloney virus-induced lymphoma (YWA): in vitro generation of cytotoxic lymphocytes

A Moloney leukemia virus-induced lymphoma of the A.SW strain, YWA, was used to generate cytotoxic cells in vitro. Cocultivation of spleen cells from in vivo primed syngeneic and semisyngeneic mice with X-irradiated YWA tumor cells for 5 days resulted in a strong killing activity against YWA. The cytotoxicity was H-2 restricted and mediated by Thy-1.2-positive lymphocytes. F1 hybrids with variable degrees of natural resistance to the YWA tumor in vivo all generated cytotoxic cells after secondary stimulation in vitro but showed differences in optimal responder:stimulator requirements.     

H-21-linked control of immunological resistance to viral leukemogenesis as a response to preleukemic cells

The mechanism of resistance to leukemogenesis by two radiation leukemia virus variants, A-RadLV and D-RadLV, was investigated. Resistance to these viruses is linked to H-21 in both B10.S and C57BL/10 mice. The resistance of virus-infected mice to transplantation of syngeneic. A- or D-RadLV-induced lymphoma cells was similar to their resistance to leukemogenesis by the same viruses. This resistance could be transferred by lymphoid cells from immune donors to normal recipients, and it was specific for RadLV lymphomas. Virus-primed (responder x sensitive)F1 hybrids rejected only resistant-type parental lymphoma cells. Hence, it appears that H-21-linked resistance to RadLV leukemogenesis is regulated by Ir genes. Resistant mice immunized by A- or D-RadLV rejected syngeneic lymphoma cells, irrespective of whether they were sensitive or resistant to the RadLV variant used for the induction of the lymphoma cells. It follows that resistant and sensitive type lymphomas are antigenically similar for the effector mechanism, and that the Ir genes may be expressed in the sensitization phase of the reaction. In virus-infected mice which are resistant to A- or D-RadLV we were able to demonstrate the presence of preleukemic lymphocytes. Normal mice could be immunized by these preleukemic cells against lymphoma challenge. These data are interpreted to suggest that mice having H-21-linked resistance to RadLV infection may be sensitized by their preleukemic cells, and that these preleukemic cells are then arrested in their development as a result of the immune response.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.     

IFN-gamma- and cell-to-cell contact-dependent cytotoxicity of allograft-induced macrophages against syngeneic tumor cells and cell lines: an application of allografting to cancer treatment.

In allogeneic tumor or skin transplantation, the rejection process that destroys the allogeneic cells leaves syngeneic cells intact by discrimination between self and nonself. Here, we examined whether the cells infiltrating into the allografts could be cytotoxic against syngeneic immortal cells in vitro and in vivo. The leukocytes (i.e., macrophages (Mphi; 55-65% of bulk infiltrates), granulocytes (20-25%), and lymphocytes (15-20%)) infiltrating into allografts, but not into autografts, in C57BL/6 mice were cytotoxic against syngeneic tumor cells and cell lines, whereas the cytotoxic activity was hardly induced in allografted, IFN-gamma-/- C57BL/6 mice. Among the leukocytes, Mphi were the major population of cytotoxic cells; and the cytotoxic activity appeared to be cell-to-cell contact dependent. When syngeneic tumor cells were s.c. injected into normal C57BL/6 mice simultaneously with the Mphi-rich population or allogeneic, but not syngeneic, fibroblastic cells, tumor growth was suppressed in a cell number-dependent manner, and tumor cells were rejected either with a Mphi:tumor ratio of about 30 or with an allograft:tumor ratio of approximately 200. In the case of IFN-gamma-/- C57BL/6 mice, however, the s.c. injection of the allograft simultaneously with tumor cells had no effect on the tumor growth. These results suggest that allograft or allograft-induced Mphi may be applicable for use in cancer treatment and that IFN-gamma induction by the allograft may be crucial for the treatment.     

Lymphokine-activated tumor inhibition in vivo. I. The local administration of interleukin 2 triggers nonreactive lymphocytes from tumor-bearing mice to inhibit tumor growth

CE-2 is a chemically induced tumor of low immunogenicity in syngeneic BALB/c mice. Nylon wool columns eluting lymphocytes from the spleen of mice bearing clinically evident (5-mm mean diameter) CE-2 tumors (CE-2 TB lymphocytes) do not react with CE-2 cells in vitro, nor are they able to affect their growth in vivo in a Winn-type neutralization assay at 5:1 lymphocyte:tumor cell ratio. However, they become able to inhibit CE-2 tumor growth when 20 U of interleukin 2 (IL 2) in 0.4 ml are injected daily for 10 days at the challenge site. In contrast, mice injected with CE-2 cells and IL 2 only display tumor takes and growth that are not significantly different from those in controls challenged with CE-2 cells alone. This lymphokine-activated tumor inhibition (LATI) is not a peculiarity of the CE-2 tumor-host combination, because different tumors can be inhibited in this way and various TB lymphocytes can initiate it. In these experiments, IL 2-rich 25,000 to 30,000 m.w. fractions were obtained routinely from the culture supernatants of a clone of EL-4 thymoma stimulated with phorbol myristic acetate. Equally active IL 2-rich preparations were obtained from rat spleen cells stimulated with concanavalin A, or from MLA 144 gibbon lymphosarcoma spontaneously releasing IL 2. Treatment of CE-2 TB lymphocytes with various antibody and C, with 2000 rad gamma-irradiation, or fractionation on Percoll density gradients suggested that radioresistant functions of Thy-1.2+, Lyt-1.2+, Lyt-2.2- and of asialo GM1+ cells are independently involved in LATI induction. These lymphocytes inhibit tumor growth by recruiting the radiosensitive effector mechanisms of the recipient mice required for ultimate tumor destruction. CE-2 tumor inhibition by LATI leaves a specific delayed-type hypersensitivity and an immunologic memory, resulting in rejection of a second lethal CE-2 challenge in a significant number of mice.     

Suppression of the in vitro secondary response to syngeneic tumor and of in vivo tumor therapy with immune cells by culture-induced suppressor cells.

Mice with advanced disseminated syngeneic tumor can be successfully treated with a combination of chemotherapy and adoptively transferred syngeneic immune cells. We have previously demonstrated that in vivo primed cells secondarily sensitized in vitro became more effective in tumor therapy, whereas primed cells cultured for 5 days without tumor stimulation became less effective than an equal number of uncultured fresh primed cells. Therefore, we examined stimulated and unstimulated cultures of tumor-primed cells for the presence of culture-induced suppressor cells, and determined whether in vivo tumor therapy with immune cells could be inhibited by concurrent inoculation of immune effector cells and cultured normal spleen cells, which contain culture-induced suppressor cells but are devoid of additional effector cells. The in vitro primary allogeneic response was suppressed by cultured normal spleen cells, or tumor-primed spleen cells previously cultured for 5 days with or without tumor stimulation. In vitro secondary sensitization to syngeneic tumor was suppressed by normal or tumor-primed cells that had previously been cultured for 5 days without stimulation. The majority of this suppression was mediated by T cells in the cultured populations. The efficacy of fresh tumor-primed cells, as well as primed cells secondarily sensitized in vitro, in adoptive chemoimmunotherapy of advanced tumor was diminished by concurrent inoculation of cultured normal cells. The cells mediating suppression of in vivo therapy required previous in vitro culture for induction, and were radiation sensitive.     

Cell-mediated immune response to syngeneic UV induced tumors: I. The presence of tumor associated macrophages and their possible role in the in Vitro generation of cytotoxic lymphocytes

A primary in vitro sensitization system employing a chromium release assay was utilized to investigate reactivity of murine spleen cells toward syngeneic ultraviolet (uv) light induced fibrosarcomas. These tumors are immunologically rejected in vivo when implanted into normal syngeneic mice but grow progressivly when implanted into syngeneic mice that had previously been irradiated with subcarcinogenic levels of uv light. Following appropriate sensitization, spleen cells from both normal and uv irradiated mice are capable of developing cytotoxic lymphocytes in vitro against the uv induced tumors. It was subsequently discovered that in situ uv induced tumors all contained macrophages of host origin that became demonstrable only after enzymatic dissociation of the tumor tissue. These macrophages were immunologically active in vitro as their presence in the stimulator cell population was necessary to achieve an optimum anti-tumor cytotoxic response following in vitro sensitization. Anti-tumor reactivity generated by mixing spleen cells and tumor cells in the absence of tumor derived macrophages could be greatly enhanced by the addition of normal syngeneic peritoneal macrophages. When in vitro anti-tumor reactivity of spleen cells from normal and uv treated mice was compared under these conditions we again found no significant difference in the magnitude of the responses. In addition, the cytotoxic cells generated in response to uv induced tumors appeared to be highly cross reactive with respect to their killing potential. Cross reactive killing was observed between all uv induced tumors tested as well as with a syngeneic benz[a]pyrene (BP) induced fibrosarcoma. No cytotoxicity was observed against normal syngeneic PEC's even through these cells were shown to be susceptible to lysis by anti-H-2^k effector cells. It was concluded that: (a) A significant number of host-derived macrophages are present in uv tumor tissue. (b) These macrophages are important for the in vitro generation of tumor specific cytotoxicity. (c) Spleen cells from uv treated mice are capable of recognizing and responding against uv tumor associated antigens in vitro. Cytotoxic effector cells generated in response to uv induced tumors appear to have specificity for tumor associated antigens (TAA) present on all uv tumors tested as well as a syngeneic BP induced tumor. The relationship between in vivo and in vitro reactivity against uv tumors is discussed.     

Splenic suppressor macrophages induced in mice by injection of Corynebacterium parvum.

Spleen cells from C57BL/6N mice injected with killed Corynebacterium parvum (CP) had a marked growth inhibitory effect on the in vitro proliferation of RBL-5 murine lymphoma cells. It was most marked 12 to 14 days after injection and was usually no longer detectable later than 21 days. It could be demonstrated at effector cell to target ratios between 20:1 and 5:1 at which normal spleen cells had a growth-promoting effect. Addition of CP to an in vitro mixture of spleen cells and tumor cells augmented the inhibitory effect of spleen cells from CP-injected mice although it conferred no inhibitory potential on normal spleen cells. Growth inhibiton by CP spleen cells was not mediated by T cells and various depletion experiments suggested that the effector cells of the phenomenon were macrophages. Spleen cells of CP-injected mice also showed strongly depressed responses to the T cell mitogens PHA and Con A and suppressed the mitogen responses of syngeneic normal spleen cells. The characteristics of the suppressor cells mediating this effect appeared to be very similar to those inhibiting lymphoma cell growth. The responses to LPS were also strongly suppressed in mice injected with 2.1 mg of CP. However, after injection of one-tenth of the dose a relative sparing of the LPS response was noted, whereas the PHA response was still suppressed.