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.     

Suppressor cell activity in mice bearing a progressively growing Simian virus 40-induced sarcoma

Summary General and cell-mediated immunity (CMI) were investigated in BALB/c mice bearing progressively growing Simian virus 40-induced (mKSA) sarcoma by means of the Winn tumor cell neutralization (WN), ¹²⁵I isotopic footpad (IFP), lymphoproliferative (LP) and plaque-forming cell (PFC) assays. Correlation between depressed antitumor immunity and the IFP responses was observed in tumor-bearing (TB) mice. Depressed LP responses to both T- and B-cell mitogens were observed in both early and late stages of tumor growth. Results obtained with the PFC assay similarly demonstrated depressed humoral immunity to sheep red blood cells (SRBC). Suppressor cell activity was demonstrated in cocultivation experiments in which spleen cells of TB mice were mixed with normal spleen cells. Treatment of TB spleen cells by passage through Sephadex G-10 columns or incubation on plastic surfaces to deplete the adherent cells restored LP responses. Cocultivation of Sephadex G-10- or plastic-adherent cells from TB mice with normal spleen cells significantly reduced mitogen-induced LP responses of normal cells. Examination of cell surface markers indicated an increase in the proportion of spleen cells bearing Fc receptors, which correlated with progressive mKSA tumor growth. There was also a correlation between Fc receptor-bearing spleen cells and macrophages, as shown by nonspecific esterase staining. These results indicate that depressed LP and PFC responses and the appearance of suppressor cells in mKSA tumor-bearing mice parallel an impaired ability to recognize (IFP responses) and neutralize (WN responses) tumor cells.     

The cell-mediated immune response to ectromelia virus infection. I. Kinetics and characteristics of the primary effector T cell response in vivo.

The cell-mediated immune (CMI) response to ectromelia virus infection in mice was studied. Virus doses from 4 × 10² up to 5 × 10⁴ PFU of an attenuated strain inoculated intravenously (iv) all induced cytotoxic T cell responses in the spleen as measured in a ⁵¹Cr release assay using virus-infected target cells. Higher virus doses gave larger responses. There was little variation between individual animals, and mice ranging in age from 4–22 weeks gave similar responses. Following iv infection, virus grew logarithmically in spleen for 2 days, then titers declined to undetectable levels by day 5. The peak of the virus-specific cytotoxic T cell response occurred at 5–6 days post-infection, as determined by calculation of effector units based on a linear log-log relationship between killer cells added and targets lysed. T cells responsible for virus clearance in vivo gave similar kinetics, suggesting the possibility that both functions are mediated by the same T cell subset. Two other categories of cytotoxic activity were also generated at low levels in the spleen during ectromelia infection or during infection with a bacterium, Listeria monocytogenes. These activities were significantly sensitive to anti-δ and complement treatment, suggesting T cell dependence, but participation of other mechanisms has not been rigorously excluded. One category lysed allogenic target cells and reached a peak at 4 days post-infection. The other lysed H-2-compatible cells, syngeneic embryo cells, and some syngeneic tumor cells but not syngeneic macrophages, and was present at similar low levels through days 1–4. These different kinetics and evidence from “cold” target competition experiments suggested that the total cytotoxic activity of immune spleen cell populations was a composite of the activities of separate cellular subsets (probably mainly T cells), killing of any one target cell type being the responsibility of a subset with receptors at least partly specific for antigens on that target cell.     

Phorbol ester-induced enhancement in lytic activity of CD8+ splenic T cells from low-dose melphalan-treated MOPC-315-tumor bearers

Summary We have previously shown that while spleen cells from untreated mice bearing a large MOPC-315 tumor are not cytotoxic in vitro for MOPC-315 tumor cells, spleen cells obtained from such mice on day 7 after low-dose melphalan (l-phenylalanine mustard);l-PAM therapy exert a substantial anti-MOPC-315 cytotoxicity [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that this anti-MOPC-315 lytic activity is evident by day 5, and peaks on day 7 after the low-dose chemotherapy, at a time when the mice are actively engaged in tumor eradication. Short-term exposure of spleen cells from mice bearing a MOPC-315 tumor and treated with low-dosel-PAM (l-PAM TuB mice) to phorbol 12-myristate 13-acetate (PMA) was found to enhance greatly the ability of these spleen cells to lyse MOPC-315 tumor cells. The highest level of anti-MOPC-315 cytotoxicity was obtained when spleen cells from tumor-bearing mice that had received chemotherapy 7 days earlier were exposed to PMA at a concentration of 1–10 ng/ml. The exertion of the enhanced anti-MOPC-315 lytic activity byl-PAM TuB spleen cells exposed to PMA was found to require CD8⁺, but not CD4⁺, T cells. The apparent specificity of the lytic activity exerted by the PMA-stimulatedl-PAM TuB spleen cells was illustrated not only by the inability of the spleen cells to lyse an allogeneic, antigenically unrelated thymoma (EL4), but also by their relatively weak lytic activity for two antigenically related syngeneic plasmacytomas. In addition, when EL4 target cells were admixed with MOPC-315 tumor cells, the lytic activity triggered in thel-PAM TuB spleen cells by the MOPC-315 tumor cells plus PMA was not effective in lysing the antigenically unrelated target cells. Moreover, even in the presence of the calcium-specific ionophore, ionomycin,l-PAM TuB spleen cells exposed to PMA were unable to lyse the EL4 target cells. Thus, fresh CD8⁺ splenic T cells froml-PAM TuB mice that are in the process of eradicating a large MOPC-315 tumor as a consequence of low-dosel-PAM therapy can be triggered with PMA to exert enhanced lytic activity against MOPC-315 tumor cells. Since the curative effectiveness of low-dose chemotherapy for MOPC-315 tumor-bearing mice requires the participation of CD8⁺ T cells that exploit a cytotoxic T lymphocyte type lytic activity for tumor eradication, it is feasible that in some situations PMA-like stimulants could be used to augment the antitumor cytotoxic activity of the CD8⁺ T cells, which in turn could improve the therapeutic outcome of low-dose chemotherapy.Supported by research grant IM-435A from the American Cancer Society and research grant B-8806 from the Bane EstateIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of Career Development Award CA-01 350 from the National Cancer Institute     

Tumor-induced alteration in macrophage accessory cell activity on autoreactive T cells

Using a tumor-model system, differences in the accessory cell capabilities on autoreactive T cells of splenic macrophages from normal and tumor-bearing hosts (TBH) were assessed in the syngeneic mixed lymphocyte reaction. Tumor development caused a drop in autoreactivity. At 0 and 7 days of tumor growth, no drop in reactivity occurred when TBH macrophages were used as accessory cells and L3T4+ autoreactive T cells from normal mice were used as responder cells. However, by day 14, there was a 32% drop in reactivity, and by day 21 only 22% of the T cell reactivity remained when TBH macrophages were used as accessory cells. Alterations in macrophage Ia antigen during tumor growth were first investigated as the potential cause of reduced autoreactivity. Before tumor growth (day 0) 59% of the splenic macrophages were found to be Ia+. Day-7 TBH macrophages showed no difference in Ia antigen expression when compared to day 0 macrophages. However, by day 14, TBH macrophages showed a 9% decrease, and by day 21 they showed a 36% decrease in the number which were Ia+. Concomitant with the decrease in the number of Ia+ cells was a decrease in the density of Ia antigen expression on day-14 and -21 TBH macrophages. In day-14 and -21 TBH macrophages, two populations were seen that were Ia+. The first had a 10%-20% decrease in Ia antigen expression per cell while the second population had a greater than 50% drop in Ia antigen expression per cell. By titrating and mixing TBH macrophages with normal host macrophages, we assessed whether they could actively mediate suppression of autoreactive T cells. A titratable suppressive phenomenon was demonstrated using day-21 TBH macrophages. In contrast, day-7 and -14 TBH macrophages titrated with normal host macrophages had no effect on the syngeneic mixed lymphocyte reactivity. Lastly, we investigated whether the macrophage-mediated suppression was caused by increased prostaglandin secretion. Addition of indomethacin to cultures increased autoreactive T cell reactivity stimulated by normal or TBH macrophages (59% and 99% increase, respectively). Although indomethacin reduced suppression mediated by TBH macrophages, autoreactivity did not return to levels induced by untreated or indomethacin-treated cells from a normal host. Taken together, the data suggested that tumor growth modulates the function of macrophage accessory cells with autoreactive T cells in at least two ways: by decreasing Ia antigen expression and by increasing suppressor activity.     

Pronounced antitumor effect of LAK-like cells induced in the peritoneal cavity of mice after intraperitoneal injection of OK-432, a killed Streptococcal preparation

Summary More than 80% of BALB/c mice bearing BAMC-1 ascites tumor were completely cured after five consecutive (once every 2 days) i. p. injections of a 0.1 mg dose of OK-432, beginning on day 2 after tumor implantation. The antitumor effect of OK-432 was abolished in athymic nu/nu mice and in anti-thymocyte globulin-treated euthymic BALB/c mice, so although OK-432 treatment did increase the length of survival, all animals eventually died as a result of tumor growth. When peritoneal exudate cells (PEC), obtained on day 12 from OK-432-treated BAMC-1-bearing euthymic mice were evaluated for in vivo tumor neutralization activity, all mice receiving an i. p. injection of the admixture of the nonadherent PEC (1×10⁷ cells) with BAMC-1 cells (1×10⁵) survived for more than 60 days. When the same nonadherent PEC (1×10⁷ cells) were i. p. transferred adoptively 1 day after the inoculation of 1×10⁵ BAMC-1 tumor cells, again all mice survived.When these in vivo active PEC were tested for cytotoxicity in vitro against fresh BAMC-1 tumor cells, natural killer (NK) sensitive syngeneic RL 1, NK-sensitive allogeneic YAC-1 cells, NK-resistant syngeneic Meth-A cells, allogeneic tumor cells (EL4, B16, and P815) and xenogenic human cells, the PEC were found to be capable of lysing BAMC-1 tumor cells together with almost all of the other tumor cells, including NK-resistant cells. Nonadherent PEC contained at least two subpopulations of killer cells. One, directed to syngeneic BAMC-1 cells, was both Thy1.2 and asialo GM1 positive, and another, directed to allogeneic YAC-1 cells, was asialo GM1 positive but Thy1.2 negative. A cold target inhibition assay also suggested the presence of more than two subpopulations.These results indicate that T cells play a determined role in the immunotherapeutic effect of OK-432 on BALB/c mice bearing BAMC-1 tumor, although the participation of activated macrophages could not be excluded. The cells responsible for killing BAMC-1 and other tumor cells appearing in the PEC on day 12 were characterized as containing at least two kinds of lymphokine-activated killer cells.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses

Summary In the present study we investigated some of the physicochemical properties of macrophage-activating factor(s) (MAF) produced by the tumor-immune Lyt-1⁺2^– T cell subset. Supernatant from mixed culture of spleen and lymph node cells, obtained from C3H/HeN mice immunized with syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma, with the corresponding tumor cells exhibited the capability of activating peritoneal exudate macrophages to exert their cytostatic and cytolytic activities on tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 antibody plus complement (C) before culturing. Anti-Lyt-2.1 and/or anti-asialo GM1 plus C treatment, however, had only marginal effect on the generation of MAF by these cells, despite the complete disappearance of natural killer (NK) cell activity of spleen and lymph node cells after the treatment with anti-asialo GM1 plus C. Thus, the tumor-specific Lyt-1⁺2^– T cell subset could fulfill a crucial role in generating MAF without the support of NK cells. The MAF activity was heat, acid, and trypsin sensitive. On Sephacryl S-300 column, MAF activity was eluated in a broad single peak around a molecular weight (m.w.) of 70,000 daltons. Antiviral activity was detected in the concentrated pool of MAF-containing fractions from Sephacryl S-300. Gel permeation analysis using HPLC also showed a coincident peak of MAF and antiviral activities at a m.w. of approximately 70,000 daltons. In addition, MAF activity was almost completely neutralized by incubation with rabbit antiserum against recombinant murine -interferon (IFN). Taken together, these results indicate that MAF generated by tumor-immune Lyt-1⁺2^– T cell subset is closely related to IFN.     

Modulation of the immune response to tumors by a novel synthetic compound,N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide (CL 259,763)

Summary CL 259,763, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide, is an orally active compound capable of modifying the reactivity of certain lymphoid cell populations affected by the growth of a tumor. The compound augmented the response of lymphocytes from tumor-primed animals to syngeneic tumor cells, resulting in a marked increase in tumor cell destruction. Likewise, it enhanced macrophage inhibitory effects on the growth of tumor cells in vitro. These activated macrophages were detectable in peritoneal exudates of treated mice 4 to 12 days after receiving a single oral dose of CL 259,763, with peak activity being demonstrable by day 7. The compound also restored the alloreactivity of lymphocytes from immunodepressed mice bearing the Lieberman plasma cell tumor, possibly by interferring with suppressor cells. Macrophages and lymphocytes from treated mice released significantly more IL-1 and IL-2-like factors in culture than did the control counterparts. Sera from treated mice also possessed more colony stimulating factor than those from normal mice. Immunoadjuvant effects were evident when the compound was administered with an inactivated L1210 leukemia vaccine and it enhanced the effectiveness of cytotoxic chemotherapy when given to mice challenged with P388 murine leukemia. These immunomodulating effects of CL 259,763 may hopefully be exploited in efforts to augment the immune response of the host to a progressively growing tumor.     

Tracking the Fate of Stem Cell Implants with Fluorine-19 MRI

Background

In this study we used cellular magnetic resonance imaging (MRI) to detect mesenchymal stem cells (MSC) labeled with a Fluorine-19 (¹⁹F) agent. ¹⁹F-MRI offers unambiguous detection and in vivo quantification of labeled cells.

Methods

We investigated two common stem cell transplant mouse models: an immune competent, syngeneic transplant model and an immune compromised, xenograft transplant model. ¹⁹F labelled stem cells were implanted intramuscularly into the hindlimb of healthy mice. The transplant was then monitored for up to 17 days using ¹⁹F-MRI, after which the tissue was excised for fluorescence microscopy and immunohistochemisty.

Results

Immediately following transplantation, ¹⁹F-MRI quantification correlated very well with the expected cell number in both models. The 19F signal decreased over time in both models, with a more rapid decrease in the syngeneic model. By endpoint, only 2/7 syngeneic mice had any detectable ¹⁹F signal. In the xenograft model, all mice had detectable signal at endpoint. Fluorescence microscopy and immunohistochemistry were used to show that the ¹⁹F signal was related to the presence of bystander labeled macrophages, and not original MSC.

Conclusions

Our results show that ¹⁹F-MRI is an excellent tool for verifying the delivery of therapeutic cells early after transplantation. However, in certain circumstances the transfer of cellular label to other bystander cells may confuse interpretation of the long-term fate of the transplanted cells.     

Cellular interactions in lymphocyte proliferation: effect of syngeneic and xenogeneic macrophages.

Secondary cell-mediated responses to ectromelia virus infection were studied using an in vitro system. Lymphoid “responder” cells from mice which had recovered from intravenous primary infection at various times prior to sacrifice, were cultured with syngeneic, virus-infected macrophages or spleen cells as “stimulator” cells at 39 °C, a temperature which prevented the virus from exerting cytopathic effects against responder cells. This restrictive temperature and medium with 2-mercaptoethanol at 10^?4M often gave viable cell yields of more than 100% of the original responder cells over 4 days of culture. Preliminary experiments showed that spleen cells from primed mice, cultured with syngeneic, infected spleen cells from normal mice gave the most powerful secondary cytotoxic cell responses as measured by ⁵¹Cr release from virusinfected H-2-compatible target cells. The cytotoxic cells were sensitive to anti-θ and complement treatment and lysed H-2-compatible, virus-infected target cells much more efficiently than infected, allogeneic target cells, thus indicating that they were T cells. Some activity against uninfected H-2-compatible target cells was also generated, but this was largely independent of the presence of virus-induced antigen, (i.e. infected stimulator cells were unnecessary) and therefore seemed to be a consequence of the cultural conditions. Cold target competition showed that this activity was the responsibility of a T cell subset separate from the virus-specific cytotoxic T cells. The peak of cytotoxic activity against virus-infected targets occurred at 4 days of culture and DNA synthesis was maximal on day 3. The concentration of cytotoxic T cells at the peak was eight-fold higher than at the peak of the splenic primary response in vivo, Memory T cells (precursors of secondary cytotoxic T cells) appeared in spleen within 12–14 days of primary infection in vivo, reached a plateau at 5–6 weeks and persisted for at least 16 months. Spleen cells appeared partly refractory to secondary stimulation in vitro at 8–10 days post-priming. This did not seem to be due to cellular migration from spleen to lymph nodes or peritoneal cavity, but its cause was not determined. Primary responses in vitro were not detectable under conditions optimal for secondary responses, thus suggesting a major quantitative, or qualitative difference between virgin and memory T cells.     

Induction of tumor-specific T lymphocyte responses in vivo by prothymosin α

We have recently reported that administration of Pro T to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunother 38:281]. In this report we demonstrate that Pro T , when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumorspecific CD8⁺ cytotoxic T lymphocytes (CTL). The Pro T -induced CD8⁺ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2K^d allelic product could inhibit the cytotoxic response. Mice receiving only Pro T developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving Pro T and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The Pro T -induced CD8⁺ CTL activity was regulated by Pro T -induced L1210-specific syngeneic CD4⁺ cells. This was shown in two different ways: first, CD8⁺-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4⁺ cells and, second, CD4⁺ cells from mice that had received both Pro T and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8⁺ cells from mice that had received only L1210 cells. Our data suggest that Pro T is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since Pro T may prove to be useful in clinical protocols aimed at cancer immunotherapy.This work was supported by a CEC grant to Dr. M. Papamichail     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

Evidence for cytostatic T cell activity in the effector mechanism against syngeneic TMT mammary tumor cells in mice

The effector mechanism of immune spleen cells against syngeneic TMT mammary tumor cells was analyzed in vitro. C3H/He mice were first inoculated with TMT tumor cells, and then the tumors were x-irradiated with 2000 rad 1 wk after the inoculation. Spleen cells from these treated mice inhibited the growth of tumor cells in vitro when assessed by (3H)-TdR incorporation by tumor cells (cytostatic activity). The same spleen cells did not have any cytotoxic activity on TMT tumor cells detected by a 51Cr-release assay. The cytostatic activity was mediated by Lyt-1+23- T cells. The purified T cells alone could not inhibit the growth of tumor cells, but accessory cells were required for the induction of cytostatic T cell activity. The accessory cells were Ia-positive, macrophage-like adherent cells. Furthermore, both T cells and macrophages were also required for the inhibition of tumor growth even after the spleen cells were activated in vitro. These results suggest T cells and macrophages play an important role in the effector mechanism against TMT mammary tumor cells. The mechanism of cytostasis by T cells and macrophages was discussed from the standpoint of the cellular interaction.     

The requirement of Ia-positive accessory cells for the induction of tumor-specific cytotoxic T lymphocytes in vitro

The mechanism for the induction of cytotoxic T cells specific for tumor-associated antigens was studied by using fractionated responder T cells, tumor cells, and accessory cells in vitro. The tumor-specific cytotoxic T cells were induced by culturing immunized spleen cells with the tumor cells in vitro for 5 days. Nylon-column-purified T cells alone did not induce cytotoxic T cells upon culture with tumor cells, but the addition of normal spleen cells as accessory cells did successfully induce the cytotoxic T cells, suggesting that the presence of accessory cells is required for the activation of tumor-specific cytotoxic T cells in vitro. The accessory function was associated with spleen cell populations adhering to a plastic dish, a Sephadex G-10 column or a nylon wool column, and was sensitive to anti-Ia serum and C treatment, but was resistant to anti-Ig serum or anti-Thy 1 serum and C treatment, suggesting that the accessory cells are Ia-positive macrophages. Not only syngeneic but also allogeneic macrophages had the accessory function and the allogeneic macrophages were also Ia positive. These results suggest that Ia-positive macrophages play a crucial role in the induction of tumor-specific cytotoxic T cells in vitro. The possible role of Ia-positive accessory cells in the induction of tumor-specific cytotoxic T cells is discussed from the standpoint of cellular interactions.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus

Summary Two IgE-producing hybridomas were established from spleen cells of Balb/c mice, which had been immunized with mouse mammary tumor virus (MMTV). These IgE monoclonal antibodies (mAbs) reacted specifically with the major envelope glycoprotein (gp36) of MMTV, as established by the immunoblot assay and by passive cutaneous anaphylaxis. The effect of the IgE mAbs (produced by clone A8) on the growth of the MMTV-secreting mammary adenocarcinoma H2712 was investigated in syngeneic C3H/HeJ mice. The mice were inoculated s.c. with either 10⁵ (100 × LD₅₀) or 10⁶ (1000 × LD₅₀) tumor cells and received repeated i.p. injections of 25 µg anti-gp36 IgE mAbs at 4-day intervals for 8 weeks. This treatment prevented the development of subcutaneous tumors in 50% of the animals. Similar protection was observed when the tumor cells (10⁵/animal) were injected i.p. 4 days prior to the beginning of the i.p. treatment consisting of injections of 25 µg mAbs at 4-day intervals for 6 weeks. However, these mAbs did not protect C3H/HeJ mice against the MMTV-negative MA16/c carcinoma cells. Hence, these results support the view that IgE-mediated cytotoxic mechanisms may play an immunologically specific antitumor surveillance role and that laboratory-induced antitumor IgE mAbs have the potential of specific therapeutic agents for in vivo destruction of tumor cells.     

Antitumor response independent of functional B or T lymphocytes induced by the local and sustained release of interleukin-2 by the tumor cells

Transfection of tumor cells with a vector containing the entire coding sequence of human interleukin-2 (hIL-2) was previously shown to convert the tumorigenic murine fibrosarcoma line CMS-5 into a non-tumorigenic line. The failure of the IL-2-secreting tumor to grow in conventional (immunocompetent) mice was attributed to the activation of CD8⁺ T cells that exhibited tumor specificity and memory. In order to determine whether or not the IL-2 produced by the tumor may be activating tumor cytotoxic effector cells other than B or T cells we have repeated this study using immunodeficient SCID and SCID-beige mice as syngeneic tumor recipients. In contrast to the rapid growth of the wild-type tumor, the hIL-2-transfected cells (N2A/IL2/CMS5) did not grow, or grew more slowly and regressed, in the mice that lack functional B and T cells. The inhibition of tumor growth associated with the local release of IL-2 was reversed in mice treated with antiasialo-GM1 antibodies specific for natural killer (NK) lineage cells. In contrast to the studies with conventional mice, the IL-2-dependent effector cells in the immunodeficient mice exhibited no evidence of memory. In vitro analysis of spleen cells from tumor-bearing mice revealed the presence of effector cells able to lyse YAC-1 target cells as well as the wild-type CMS-5 and the IL-2-transfected variant tumor lines but unable to lyse P815 cells. The pattern of selective target cell killing and the kinetics of killing were indistinguishable from those observed using tumor necrosis factor (TNF) the mediator associated with natural cytotoxicity cell killing of tumor cells. Histopathology of the IL-2-secreting tumors in SCID mice reveals the presence of infiltrating lymphoid cells and macrophages that were not observed in the CMS-5 tumors. Consistent with the notion that the tumor killing in the SCID mice was mediated by TNF, mice bearing IL-2-secreting tumors had elevated levels of serum TNF and little or no effector cell activity, or TNF was found in tumor-bearing mice treated with anti-asialo-GM1 antibody. The results indicate that the cytokine-induced tumor regression observed in the IL-2-transfected tumors is a more complex phenomenon than previously recognized and one that is mediated by effector cells of the NK cell and/or monocyte/macrophage lineages, in addition to CD8⁺ T cells.This investigation was supported by awards from Department of Health and Human Services, Public Health Service: CA09 581 (TA), CA25 253, CA54 491, CA57 974 and CA22 786 (RBB), and Natural Sciences and Engineering Council, Canada (BAC)     

Effects of Corynebacterium parvum on depressed immunological reactions in EL-4-tumor-bearing mice

Summary Effects of Corynebacterium parvum on the development of plaque-forming cells (PFC), cell-mediated cytotoxicity (CMC), and delayed footpad reaction (DFR) to chicken erythrocytes (CRBC) were investigated in EL-4-bearing syngeneic mice. PFC, CMC, and DFR responses after the primary immunization were suppressed in tumor-bearing mice and restored by C. parvum treatment. PFC and CMC responses in tumor-bearing mice were restored by the transfer of spleen cells of C. parvum-treated normal mice. Such powers of recovery were abrogated by the removal of glass-adherent cells but not by the removal of -positive or Ig-positive cells. DFR was suppressed not only in the primary but also in the secondary immunization, in contrast to PFC and CMC; the secondary responses of these types were not suppressed in tumor-bearing mice. Positive DFR was not elicited in tumor-bearing mice after adoptive transfer of sensitized lymphocytes from normal immune donors. The DFR became positive in such tumor-bearing recipients when they were treated by C. parvum. Macrophage functions in the induction phase of the immune response as accessory cells and in the expression of DFR as secondary cells appear to be suppressed in tumor-bearing mice and restored by C. parvum.     

Histological analysis of IL-2 induced regression of murine solid SL2-tumors

When DBA/2 mice are inoculated both intraperitoneally (i.p.) and subcutaneously (s.c.) with syngeneic SL2 lymphoma cells and treated i.p. on day 10–14 with 20,000 units IL-2/day, about 50% of the mice reject both the ascitic tumour and the s.c. tumour. During IL-2 therapy large areas of necrosis appear in the solid SL2 tumours between day 12 and 15. Immunohistochemical studies show that only a small number of infiltrating cells is present in the tumours. The percentage of macrophages (MHC-II⁺)in the tumours is about 1 and the percentage of T-lymphocytes (-TCR⁺) about 0.5. No differences in the numbers of infiltrating cells are seen in untreated and IL-2 treated tumour bearing mice. The tumoursurrounding infiltrate consists mainly of mononuclear cells: about 50% macrophages, 20% CD8⁺ cells, and 15% CD4⁺ cells. No tumour-infiltrating cells were found that express the IL-2 receptor.We conclude that direct cytotoxic activity of tumour infiltrating cells cannot account for the rapid occurrence of necrosis.When L3T4⁺ cells were eliminated by treating the mice with-L3T4 monoclonal antibodies before tumor inoculation and treatment with rIL-2, tumor eradication did not occur. So, L3T4⁺ helper T-cells are essential for IL-2-mediated tumour regression. Exogenous rIL-2 is not directly responsible for the induced tumour regression. A significant stagnation of intratumoural bloodflow is observed after histological analysis; yet it still needs to be determined whether this is the primary cause or consequence of the observed necrosis.Abbreviations BSA bovine serum albumin - CTL cytotoxic T-lymphocyte - FACS fluorescence activated cell sorter - HE haematoxylin and eosin - IFN interferon - IL-2 interleukin-2 - IL-2R interleukin-2 receptor - i.p. intraperitoneal(ly) - i.V. intravenous(ly) - LAK lymphokine-activated killer - MHC major histocompatibility complex - PBS phosphate buffered saline - s.c. subcutaneous(ly) - TCR T-cell receptor - TNF tumour necrosis factor