Cytotoxic T lymphocytes in DBA/2 mice harboring L5178Y cells in a tumor-dormant state

Summary Previous experiments have demonstrated a temporal relationship between the decline of cytotoxic T lymphocyte (CTL) activity in the peritoneal cavity of DBA/2 mice harboring L5178Y cells in a tumor-dormant state and the appearance of ascitic tumors. Some tumor-dormant mice remain clinically normal for many weeks after the decline of CTL activity, and this activity can be rapidly restimulated by an IP inoculation of irradiated L5178Y cells. We report here that the peritoneal cells from many tumor-dormant mice can be stimulated to cytolytic activity in vitro when cultured for 4 days either with or without the addition of irradiated L5178Y cells. Peritoneal cell populations which cannot be stimulated in vitro can suppress the generation of CTL in those populations which can be stimulated. The tumor-dormant state may terminate when suppressor cells in the peritoneal cavity of tumor-dormant mice inhibit the generation of CTL activity and permit tumor cells to produce an ascitic tumor. Abbreviations used in this paper: C, complement; CTL, cytotoxic thymus-derived lymphocyte; PC, peritoneal cells; DPC, days post challange; NAD, nonadherent; SC, subcutaneous; IP, intraperitoneal     

Synergistic cytolytic activity by combined populations of peritoneal T lymphocytes and macrophages during the L5178Y cell tumor-dormant state in DBA/2 mice

It was previously reported that the establishment of the L5178Y cell tumor-dormant state in DBA/2 mice is mediated principally by a peritoneal cytolytic T-cell response that reaches peak levels 4 days after L5178Y cell challenge, lyses more than 99% but less than 100% of peritoneal L5178Y cells, and gradually wanes to background levels by 40–70 days postchallenge (DPC). At this time the majority of mice are clinically normal, and contain a relatively small number of L5178Y cells in the peritoneal cavity. During the tumor-dormant state, mice that harbor more than 10⁴ L5178Y cells contain peritoneal macrophage-mediated cytolytic activity. We report here that tumor-dormant mice that contain fewer than 10⁴ peritoneal L5178Y cells also produce cytolytic activity in vitro, but that it is synergistic, in that the cytolytic activity of adherent (AD) peritoneal cells (PEC) and nonadherent (NAD) PEC cultured together is greater than the additive lysis produced by these cell populations when cultured separately. This synergistic cytolytic activity is: (1) effector cell density dependent, (2) dependent on the tumor-dormant status of the NAD and AD PEC donor mice, (3) protracted in its kinetics during a 48-hr in vitro assay, and (4) dependent on an interaction between NAD T cells and AD phagocytic macrophages. The consistent detection of this in vitro-assayed cytolytic activity in PEC of tumor-dormant mice which harbor small endogenous tumor burdens suggests that it reflects an in vivo cytotoxic effector mechanism involved in the long-term maintenance of the tumor-dormant state.     

Immune regulation of the L5178Y murine tumor-dormant state. II. Interferon-gamma requires tumor necrosis factor to restrain tumor cell growth in peritoneal cell cultures from tumor-dormant mice

L5178Y lymphoma cells are restrained from progressive growth in peritoneal cell ("in vitro tumor-regressor" PC) cultures prepared from many DBA/2 mice which harbor the tumor cells in the peritoneal cavity in a tumor-dormant state. Treatment of these PC cultures with 'antibodies to murine interferon-gamma (MuIFN-gamma) and murine tumor necrosis factor (MuTNF) but not with antibody to interleukin 2 (IL-2) receptors eliminated the restraint on tumor cell growth and permitted their progressive proliferation. L5178Y cells were found to be resistant to the direct toxic effects of large concentrations (3,000 U/ml) of MuIFN-gamma and of MuTNF, either alone or in combination. Treatment of PC cultures from tumor-dormant mice, in which tumor cells grew progressively ("in vitro tumor-progressor"), but not PC cultures from normal mice, with exogenous MuIFN-gamma resulted in a marked inhibition of tumor cell growth. The MuIFN-gamma-induced cytotoxic activity was cell-mediated since no soluble tumor-cytotoxic factors could be detected in the cultures. MuIFN-gamma induced cytotoxic activity in plastic-adherent peritoneal cell (AD-PC) cultures, but induced no cytotoxic activity in nonadherent-PC cultures unless small numbers (2%) of AD-PC were present, and inclusion of antibody to MuTNF in these mixed PC cultures blocked the development of cytotoxic activity. Antibody to MuTNF also blocked the development of cytotoxic activity in cultures of MuIFN-gamma-treated whole PC and AD-PC from tumor-dormant mice. These results indicate that MuIFN-gamma and MuTNF are both important in restraining tumor cell growth in PC cultures from tumor-dormant mice, and that MuIFN-gamma requires the presence of MuTNF to induce cytotoxic activity in these cultures.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Evidence for interaction between T cell populations of tumor-bearing and normal mice in immune suppression

Spleen cells of DBA/2 mice bearing subcutaneous implants of the syngeneic tumor L5178Y induce suppression of the in vitro antibody response of normal spleen cells to sheep erythrocytes (SRBC). Cells mediating suppression are detected in the spleens of tumor-bearing mice as early as 24 hr post-implantation but are no longer detected there 15 days post-implantation. These spleen cells are nylon wool nonadherent, sensitive to anti-Thy 1.2 + C and anti-Lyt 1.1 + C, and insensitive to anti-Lyt 2.1 + C treatment. The anti-SRBC response of the unfractionated spleen cells from the tumor-bearing mice is not itself suppressed at the cell numbers used. This along with the finding that suppression occurs in the presence of spleen cells from normal mice suggest that a cell population from the normal mouse spleen is also involved in the suppression. Spleen cells from mice inoculated with irradiated (nonproliferating) L5178Y cells are similarly capable of mediating nonspecific suppression for the same limited period of time after the inoculation. In addition, spleen cells from mice stimulated with several nontumorigenic cellular antigens interact with normal spleen cells to produce suppression. These findings suggest that suppression observed in vitro with spleen cells from these tumor-bearing mice may be the result of antigen-activated cells triggering normal immunoregulatory cells.     

Secondary in vitro generation of CTL specific for MM antigen by stimulation with somatic tumor hybrid, but not with parental tumor cells

Somatic tumor hybrid cells (L-FM3A#2), obtained by hybridization of MM tumor cells (FM3A/R) with HGPRT-less L cells, could induce CTL directed against MM antigen, a tumor-associated transplantation antigen that is expressed on some ascitic mammary tumor cell lines of C3H/He mice; parental tumor cells (FM3A/R) could not produce such CTL in syngeneic mice. In this study, the mechanisms of the generation of CTL by stimulation with L-FM3A#2 hybrid cells were investigated. In the secondary in vitro stimulation system, L cell component(s) that were introduced into hybrid cells by cell fusion play a role in the induction of CTL, as shown by the fact that stimulation with a mixture of L and FM3A/R cells could induce MM antigen-specific CTL, and that the killer helper effect of L cells could be replaced by cell free culture supernatants obtained from co-cultures of unprimed C3H spleen and L cells. IFN activity, but no IL 2 activity, was detected in the culture supernatants. Both IFN and killer helper activity were lost with pH 2 treatment; furthermore, CTL were generated by stimulation of primed spleen cells with FM3A/R cells in the presence of mouse beta-IFN, but not in its absence. These results suggest that IFN liberated by the helper cells that recognize L cell component(s) on the surfaces of tumor hybrid cells plays an essential role in the generation of CTL specific for MM antigen.     

Limited importance of CD40/CD40L interaction in the B7-dependent generation of anti-MOPC-315 cytotoxic T lymphocyte activity by tumor bearer splenic cells stimulated in vitro in the presence of tumor necrosis factor

 We have previously illustrated the importance of B7-2 expression for the enhanced generation of cytotoxic T lymphocyte (CTL) activity by stimulation cultures of tumor bearer splenic cells to which tumor necrosis factor α (TNFα) has been added. Here we show that the B7-1 molecule is also important for CTL generation by such stimulation cultures, although to a much lesser extent than the B7-2 molecule. In addition, we show the importance of CD40/CD40L interaction for the expression of the B7-2 molecule, but not the B7-1 molecule, by tumor bearer splenic cells stimulated in vitro in the presence of TNF. The CD40/CD40L interaction is also shown to be important for the generation of CTL activity by tumor bearer splenic cells stimulated in vitro in the presence of exogenous TNF. However, the CD40/CD40L interaction is less important for the generation of enhanced CTL activity than for the expression of an elevated level of B7-2. Specifically, blockade of CD40/CD40L interaction, which reduced the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the presence of TNF to the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the absence of exogenous TNF, failed to reduce the level of CTL generated to the level generated by tumor bearer splenic cells stimulated in the absence of exogenous TNF. Finally, blockade of CD40/CD40L interaction was inferior to blockade of B7-2/CD28 interaction in inhibiting the generation of CTL activity by tumor bearer splenic cells stimulated in the presence of exogenous TNF. Thus, although CD40/CD40L interaction is important for the generation of enhanced CTL activity by stimulation cultures of tumor bearer splenic cells to which TNF has been added, TNF also mediates its potentiating effect for CTL generation by such stimulation cultures via other mechanisms that are independent of CD40/CD40L interaction but dependent on B7-2 expression. Received: 31 December 1997 / Accepted: 27 March 1998     

In vitro expression of secondary antitumor immunity by in vivo tumor-sensitized T cells: Inhibition by tumor-induced suppressor T cells

Summary In vitro cultivation of memory immune cells from P815- or P388-immune mice with corresponding irradiated tumor cells induced generation of cytolytic T cells (CTL). The induction of CTL generation, as well as the cytolytic activity itself, was tumor-specific. The in vitro generation of CTL from P815- or P388-immune cells was suppressed by spleen cells from mice bearing corresponding progressive tumors (tumor size 15 mm). The tumor-induced suppressor cells suppressed the in vitro generation of CTL, but did not affect their cytolytic function. The suppression was tumor-specific and was mediated by Ly1⁺2^–L3T4⁺ T cells. Treatment of suppressor cell donors with cyclophosphamide or sublethal -radiation completely abolished the ability of their spleen cells to inhibit the in vitro CTL generation.     

The role of suppressor T cells in BCNU-mediated rejection of a syngeneic tumor

The present investigation was initiated to determine the mechanism by which 1,3-bis(2-chloro-ethyl)-1-nitrosourea (BCNU) treatment of tumor-bearing mice results in a high percentage of surviving mice which are resistant to subsequent homologous tumor challenge. Spleen cells from C57BL/6 mice bearing the syngeneic LSA ascites tumor failed to demonstrate significant tumor-specific cytotoxic T lymphocyte (CTL) activity when stimulated in vitro with irradiated tumor cells. This lack of CTL activity correlated with the presence and high activity of two types of CTL-regulatory suppressor T cells (Ts), tumor-specific Thy-1+, Lyt-1-2+ and tumor-nonspecific Thy-1+, Lyt-1+2+ cells, as demonstrated by a double-positive selection technique. In contrast, spleen cells from BCNU-treated tumor-bearing mice generated high tumor-specific CTL activity when stimulated in vitro with irradiated tumor cells. This CTL activity correlated with the lack of demonstrable tumor-specific Ts and greatly diminished tumor-nonspecific Ts activity. The tumor-specific helper activity of Thy-1+, Lyt-1+,2- cells was found to be similar in both BCNU-treated and untreated tumor-bearing mice. BCNU-treated mice that survived a primary LSA tumor challenge (referred to as BCNU-cured mice) resisted subsequent challenge with the homologous (LSA) but not with a heterologous syngeneic tumor (EL-4). However, rejection of a secondary challenge with LSA tumor by BCNU-cured mice was inhibited by adoptive transfer of spleen cells from either normal mice or mice bearing LSA tumors. Furthermore, LSA tumor cells that failed to evoke tumor-specific CTL activity in normal mice could induce high CTL activity in BCNU-cured mice. The present study suggests that, in addition to its direct tumoricidal activity, BCNU inhibits the induction of tumor-specific Ts, thereby explaining why a high percentage of mice survive a primary syngeneic tumor challenge after treatment with BCNU, and also resist subsequent rechallenge with the homologous tumor.     

Activity of tumor-associated lymphoid cells at short intervals after administration of irradiated syngeneic and allogeneic tumor cells.

After a single intraperitoneal injection of irradiated tumor cells, host cells capable of responding against syngeneic tumors were detected in peritoneal exudates of mice. Although irradiation of the injected tumor prevented its overgrowth, it did not significantly alter the antigenicity of the tumor. Immunologic activities of tumor-associated host cells in the peritoneal cavity were continuously monitored, starting 48 hr after tumor administration. In vitro cell-mediated lysis of syngeneic tumors appeared as early as 3 days after irradiated tumor administration. In addition, peritoneal exudate cells from inoculated mice were capable of adoptively transferring immunity. Purification of these peritoneal exudate cells on nylon wool columns yielded a nonadherent Ig-negative lymphocyte fraction whose cytolysis was tumor-specific and T cell-associated. The macrophage-free lymphocyte fraction exhibited a higher in vitro activity against tumors than unpurified peritoneal exudates. This tumor-host system allowed the study of cells which directly interact with the tumor cells in vivo, starting shortly after tumor administration. The results reported in this paper show that tumor-associated lymphoid cells capable of mounting anti-tumor response in vivo and in vitro can be demonstrated as early as 3 days after tumor inoculation.     

Characterization of L5178Y leukemic cells which rapidly develop and lose implantation ability.

Two populations of L5178Y murine leukemic cells, maintained by different methods, were studied for their implantation ability in BDF₁ mice. Implantation ability was measured by number of tumor nodules formed, liver weight, and day of death of the animal. 1) Cells from a population grown for 10 years $\text{in vitro}$ had no implantation ability; i.e., no tumor nodules were formed when injected into the tail vein. After 30 days of growth in the peritoneal cavity of BDF₁ mice, these same cells were injected into the tail vein and 10 days later had produced over 200 liver tumor nodules. When cells taken from these tumors were recultured for 60 days $\text{in vitro}$, they lost the acquired implantation ability, but regained it after another single peritoneal passage. 2) L5178Y murine leukemic cells grown for six years in ascites tumor cells were extremely tumorigenic; over 200 tumor nodules appeared in the liver after tail vein injection. These cells were not rendered less tumorigenic and did not lose their implantation ability by $\text{in vitro}$ culturing for 60 days. The results suggest that implantation ability is a property of the cell's growth environment; furthermore, they have strong implications for the $\text{in vivo}$ and $\text{in vitro}$ manipulation of this property.     

Cellular interactions in the cytotoxic T lymphocyte response to herpes simplex virus antigens: differential antigen activation requirements for the helper T lymphocyte and cytotoxic T lymphocyte precursors

The role and induction requirements of helper T lymphocyte responses to herpes simplex virus type 1 (HSV-1) was examined. Splenocytes from mice that had been primed in vivo with infectious HSV-1 can be restimulated in vitro with live or partially UV-inactivated HSV-1 to generate high levels of herpes virus-specific cytotoxic T lymphocyte (CTL) activity. By comparison, naive splenocytes or splenocytes taken from mice primed with heat-inactivated HSV-1 failed to generate CTL after in vitro viral stimulation. In addition, infectious HSV-primed splenocytes can be rendered unresponsive to secondary in vitro restimulation by pretreatment with anti-Lyt-1 antiserum plus complement. Spleen cells were taken from mice that had been primed and restimulated in vivo with infectious HSV-1. Two days after the second priming, splenocytes were prepared and irradiated. These cells were capable of assisting in the generation of CTL to varying degrees in all of the above unresponsive populations of cells. The irradiated cells did not produce detectable levels of CTL activity when cultured alone with antigen. Also, if the irradiated splenocytes were treated with anti-Lyt-1 plus complement before their addition to cultures, all restorative activity was ablated. In contrast, irradiated splenocytes from mice that had been primed and restimulated in vivo with either heat-inactivated or UV-inactivated HSV-1 were unable to provide help to naive or helper-depleted cultures. The failure to supply helper activity appears not to involve the preferential activation of suppressor cells, as evidenced by cell mixing experiments and the addition of concentrated, antigen-stimulated spleen cell supernatant fluids to secondary anti-HSV-1 splenocyte cultures. Proliferative assays using interleukin 2- (IL 2) dependent cell lines as a measure of relative helper activity indicated that the inactivated forms of HSV-1 were incapable of effectively enlisting helper activity. These experiments therefore suggest that the observed failure of heat-inactivated or UV-inactivated HSV-1 preparations to induce anti-HSV CTL responses reflects the inability of the HSV-1-specific subset of helper T lymphocytes to recognize these forms of the antigen.     

CD40 ligation restores cytolytic T lymphocyte response and eliminates fibrosarcoma in the peritoneum of mice lacking CD4+ T cells

Absence of CD4⁺ T cell help has been suggested as a mechanism for failed anti-tumor cytotoxic T lymphocytes (CTL) response. We examined the requirement for CD4⁺ T cells to eliminate an immunogenic murine fibrosarcoma (6132A) inoculated into the peritoneal cavity. Immunocompetent C3H mice eliminated both single and repeat intraperitoneal (IP) inoculums, and developed high frequency of 6132A-specific interferon-γ (IFNγ)-producing CTL in the peritoneal cavity. Adoptive transfer of peritoneal exudate cells (PEC) isolated from control mice, protected SCID mice from challenge with 6132A. In contrast, CD4 depleted mice had diminished ability to eliminate tumor and succumbed to repeat IP challenges. Mice depleted of CD4⁺ T cells lacked tumor-specific IFNγ producing CTL in the peritoneal cavity. Adoptive transfer of PEC from CD4 depleted mice failed to protect SCID mice from 6132A. However, splenocytes isolated from same CD4 depleted mice prevented tumor growth in SCID mice, suggesting that 6132A-specific CTL response was generated, but was not sustained in the peritoneum. Treating CD4 depleted mice with agonist anti-CD40 antibody, starting on days 3 or 8 after initiating tumor challenge, led to persistence of 6132A-specific IFNγ producing CTL in the peritoneum, and eliminated 6132A tumor. The findings suggest that CTL can be activated in the absence of CD4⁺ T cells, but CD4⁺ T cells are required for a persistent CTL response at the tumor site. Exogenous stimulation through CD40 can restore tumor-specific CTL activity to the peritoneum and promote tumor clearance in the absence of CD4⁺ T cells.Supported in part by grants from Children’s Hospital of Wisconsin Foundation, Society of University Surgeons Foundation, Florence and Marshall Schwid Foundation, Elsa Pardee Foundation, Kathy Duffy Fogarty Fund of the Greater Milwaukee Foundation (JS) and NIH grant RO1-CA-37156 (HS); Andrew Lodge and Ping Yu have contributed equally to this work.     

Analysis of protective and cytotoxic immune responses in vivo against metabolically inactivated and untreated cells of a mutagenized tumor line (requirements for tumor immunogenicity)

The immunogenicity of a mutagenized subline (ESb-D) of the weakly immunogenic T-cell lymphoma L 5178 Y ESb has been characterized. The injection of 10(6) ESb-D cells ip did not establish lethal tumors in untreated DBA/2 mice but established tumors in sublethally irradiated mice. Injection of ESb-D cells into otherwise untreated DBA/2 mice established also a state of protective immunity against the subsequent injection of otherwise lethal doses of ESb tumor cells. Protection was only obtained after injection of intact but not UV-irradiated or mitomycin-C-treated ESb-D cells. A direct T-cell-mediated cytotoxic activity was also demonstrable in the spleen cells of DBA/2 mice after injection of ESb-D cells but not ESb cells. The cytotoxic activity was variant specific for ESb-D target cells, and it was induced only with intact but not UV-irradiated or mitomycin C-treated ESb-D cells. This suggested that the induction of protective and cytotoxic immunity may require the persistence of the antigen or unusually high antigen doses. The in vivo priming for a secondary in vitro cytotoxic response, in contrast, was achieved with intact and also with mitomycin C-treated ESb-D cells but again not with UV-irradiated ESb-D cells. This indicated that the metabolic activity was a minimal requirement for the in vivo immunogenicity of the ESb-D tumor line. The secondary cytotoxic activity was demonstrable on ESb-D and ESb target cells and could be restimulated in vitro about equally well with ESb-D and ESb cells. But the in vivo priming was again only obtained with ESb-D cells and not with ESb cells. These experiments thus demonstrated that the requirements for immunogenicity are more stringent in vivo than in vitro, and more stringent for the induction of direct cytotoxic and protective immunity in vivo than for the in vivo priming for secondary in vitro responses.     

Augmentation of an antitumor CTL response In vivo by inhibition of suppressor macrophage nitric oxide

Evidence is provided that inhibition of macrophage NO production can augment in vivo CTL responses. Specifically, administration of NG-monomethyl-l -arginine (NGMMA) via osmotic pumps increases the tumor-specific CTL response against the P815 mastocytoma in the peritoneal cavity of preimmunized mice. Both the magnitude and duration of the CTL response were increased. That the augmented CTL response resulted from inhibition of the NO synthase pathway is supported by the finding that macrophage NO production from NGMMA-treated mice was reduced. Also, in vitro inhibition of NO production by peritoneal exudate cells from P815 tumor-challenged mice augmented the secondary CTL response observed. Cell proliferation was augmented by NGMMA in these cultures, suggesting that macrophage NO may suppress CTL by inhibiting clonal expansion. NO-mediated inhibition was observed in vivo in this experimental system, even though the CTL response is not suppressed, in that tumor rejection occurs. Therefore, the present results are consistent with the conclusion that macrophage NO-mediated inhibition of the CTL response is a side effect of activating macrophages rather than resulting from the action of a distinct subset of what have long been termed suppressor macrophages. Most important, the results indicate that NO-mediated suppressor macrophage activity can be an important CTL immunoregulatory element in vivo.     

Functional activity in vivo of effector T cell populations. II. Anti-tumor activity exhibited by syngeneic anti-MoMULV-specific cytolytic T cell clones

The functional activity in vivo of murine tumor-specific cytolytic T lymphocyte populations and clones was studied. Tumor cell destruction induced after the i.v. injection of cytolytic effector cells was quantitated by monitoring the elimination of 131IUdR-labeled tumor cells in the peritoneal cavity by using whole-body counting techniques. Mixed leukocyte-tumor cell cultures were established by using spleen cells from C57BL/6 regressor mice that had rejected an intramuscular tumor induced by the injection of MSV-MoMuLV virus. This effector cell population was observed to eliminate syngeneic MoMuLV-induced tumor cells in a dose-dependent manner. Treatment of the effector cell population with monoclonal anti-Lyt-2 antibodies plus complement totally abrogated their ability to induce tumor cell destruction in the peritoneal cavity. MSV-MoMuLV-specific Lyt-2+ cytolytic T cell clones derived by micro-manipulation of T lymphocyte-tumor cell conjugates were also tested for functional activity in vivo. Several clones induced a rapid, specific elimination of 131I-labeled MBL-2 tumor cells from the peritoneal cavity after i.v. injection, whereas others were inactive. Both active and inactive clones were highly cytolytic and secreted MAF/IFN-gamma lymphokines. In contrast to previous results obtained in a tumor allograft model, the MSV-MoMuLV-specific cytolytic T cell clones that were active in vivo did not proliferate in vitro in response to stimulation with irradiated tumor cells plus filler spleen cells in the absence of an added source of interleukin 2.     

Immunomodulating activity of 1-methyl and 1-ethylascorbigens

1-Methyl- and 1-ethylascorbigens, derivatives of indole and ascorbic acid are the vitamin C depo-forms with antitumor activity. Relation between the antitumor activity of the derivatives and their immunostimulating action was studied. The derivatives showed similar properties in vitro: they stimulated lymphocyte blast transformation, insignificantly stimulated formation of cytotoxic T-lymphocytes (CTL) in the allogenic mixed culture of lymphocytes (AMLC), inhibited cytotoxicity of natural killer cells (NKC) and had no cytotoxic action in cultures of tumors CaOv and others. In vivo 1-methylascorbigen promoted an increase in the splenocyte count in mice, stimulated 16-fold generation of CTL in AMLC of the splenocytes and retarded the growth of ACATOL tumor in thymus-free mice. 1-Ethylascorbigen had no such effects. The antitumor action of 1-methylascorbigen is likely to be associated with stimulation of CTL generation and not with the increase in the activity of NKC.     

In situ activation of syngeneic tumour-specific cytotoxic T lymphocytes: Intra-pinna immunization followed by restimulation in the peritoneal cavity

Summary Tumour-specific cytotoxic T lymphocytes (CTL) are usually obtained after immunization in vivo and restimulation of immune cells in vitro. We here describe the generation of syngeneic tumour-specific CTL within no more than 9 days by priming and restimulation in vivo. This is achieved only if the correct sites are used both for primary immunization (ear pinna) and for restimulation (peritoneal cavity). The kinetics of immune T cell induction and of the secondary response in vivo will be reported. While a secondary CTL response could be generated in the peritoneal cavity, this was not possible in the spleen, no matter which routes of antigen restimulation were used. Upon transfer of immune spleen cells into the peritoneal cavity but not into the spleen, a secondary response could be generated upon in situ restimulation, indicating the importance of the correct microenvironment for this type of response. The peritoneal effector cells were true T cells and recognized a tumour-associated antigen in association with the K^d major histocompatibility (MHC class I) antigen. Finally the activated tumour-specific peritoneal exudate cells were able to transfer protective immunity without exogenous interleukin-2 into normal syngeneic mice.     

Suppressor T cells regulate the cytolytic T lymphocyte response to syngeneic tumors induced by murine sarcoma virus (MSV) in the mouse.

Studies were designed to analyze the immune activities of spleen cells from mice previously injected with murine sarcoma virus (MSV) and undergoing the processes of MSV tumor growth and rejection. Fractionation of MSV-primed spleen cells according to cell size by velocity sedimentation at unit gravity showed that MSV-specific cytolytic T lymphocytes (CTL) generated in vivo underwent an apparent transition in size from large to small cells as the tumor regressed. The majority of CTL precursors, however, were invariably recovered among small to medium-sized MSV-immune cells, as revealed to CTL generation in vitro in secondary mixed leukocyte-tumor cell cultures (MLTC). Evidence was obtained for the existence in MSV-immune spleens of two suppressor cell populations capable of inhibiting CTL generation in vitro: one population probably consisted of macrophages and could be removed by treatment with carbonyl iron; the second population was comprised of T cells and inhibited the differentiation of tumor-immune CTL precursors in a selective manner. These results provide a preliminary overview of the mechanisms regulating the generation, differentiation, and activity of tumor-specific CTL in a syngeneic model system.     

A role for mast cells and the vasoactive amine serotonin in T cell-dependent immunity to tumors

The involvement of mast cells in anti-tumor resistance was studied by employing 2 strains of mast cell deficient but otherwise immunocompetent mice on a C57BL/6 (H-2b) background (W/Wv and Sl/Sld) and their respective normal +/+ littermate controls. Sensitization of control mice with irradiated semisyngeneic B16 melanoma cells (H-2b) resulted in protection against subsequent challenge with viable B16 cells, in contrast to sensitization of either W/Wv or Sl/Sld mice. The involvement of serotonin in antitumor resistance was studied by employing 2 serotonin active drugs: reserpine, that depletes mast cells of serotonin; and methysergide, a serotonin antagonist. Sensitization of BDF1 mice with irradiated B16 cells and sensitization of DBA/2 mice (H-2d) with irradiated SL2 cells (H-2d) resulted in protection against subsequent challenge with viable B16 cells and viable SL2 cells, respectively. Treatment with either reserpine or methysergide resulted in a decreased protection. Delayed-type hypersensitivity (DTH) footpad responses to allogeneic L5178Y (H-2d) tumor cells in C57BL/6 mice showed a biphasic reaction pattern, similar to that found in DTH responses to simple reactive haptens, such as picryl chloride. Moreover, the early swelling responses were also dependent on T cells and on mast cells. BDF1 mice carrying a semisyngeneic L5178Y tumor on the chest showed an early swelling response after footpad challenge but no late response, possibly indicating that selective down regulation of the late component of DTH was associated with progressive tumor growth in these animals. The biphasic patterns of DTH to both tumor cells and picryl chloride and the T cell and mast cell dependence of both antitumor resistance and DTH to tumor cells suggest that T cell-dependent activation of mast cells to allow entry of mononuclear leukocytes into sites of tumor growth is similar to the mechanism that occurs in DTH.