Hiroto Nakajima

The mode of recognition of tumor antigens by noncytolytic-type antitumor T cells: role of antigen-presenting cells and their surface class I and class II H-2 molecules.

SummaryThe present study investigated the role of antigen-presenting cells (APC) in the activation of noncytolytic T cells against tumor antigens. The noncytolytic-type T cells exerted their antitumor effect by producing γ-interferon (IFN-γ) and by activating macrophages as the ultimate effectors. The production of IFN-γ by these noncytolytic T cells following the stimulation with tumor cells required the participation of Ia+ APC, since the depletion of APC from cultures of tumor-immunized spleen cells resulted in almost complete inhibition of the IFN-γ production. Both L3T4+ and Lyt-2+ subsets of T cells were capable of producing IFN-γ, and the requirement of APC for the production of IFN-γ was the case irrespective of whether noncytolytic T cells were of L3T4+ or Lyt-2+ phenotype. More importantly, it was demonstrated that the production of IFN-γ by L3T4+ and Lyt-2+ T cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to cultures. These results indicate that antitumor L3T4+ or Lyt-2+ noncytolytic T cells are activated for the IFN-γ production by recognizing tumor antigens in the context of self-class II or -class I H-2 molecules on APC.

Mechanisms for Recognition of Tumor Antigens and Mediation of Anti‐tumor Effect by Noncytolytic Lyt‐2+ T Cell Subset

The mode of anti‐tumor function in vivo of noncytolytic Lyt‐2+ T cells from C3H/He mice hyperimmune to syngeneic MH134 hepatoma was investigated in a double diffusion chamber system which was recently established in our laboratory. C3H/He mice were implanted intraperitoneally with the double diffusion chamber unit in which each chamber contained either L3T4+ T cell‐depleted MH134‐hyperimmune spleen cells plus mitomycin C‐treated MH134 tumor cells or other syngeneic X5563 viable tumor cells plus normal spleen cells as a source of macrophages. Inclusion of anti‐MH134 Lyt‐2+ T cells together with MH134 tumor cells in one chamber resulted in comparable growth inhibition of viable X5563 tumor cells in the other chamber to that obtained by unfractionated MH134‐hyperimmune spleen cells. The induction in the Lyt‐2+ T cell‐containing chamber of anti‐tumor effect to be delivered into the other chamber was dependent on the co‐existence of la‐positive adherent cells along with Lyt‐2+ T cells. Although adherent cell‐depleted Lyt‐2+ T cells regained the inducibility of anti‐tumor immunity when supplemented with splenic adherent cells, the addition of adherent cells pretreated with chloroquine failed to restore the ability of Lyt‐2+ T cells to induce their anti‐tumor effect. In addition, paraformaldehyde‐treated MH134 tumor cells instead of untreated tumor cells were not capable of activating Lyt‐2+ T cells. These results indicate that a portion of Lyt‐2+ T cells exerts their anti‐tumor effect by a mechanism distinct from direct tumor cell lysis and that their activation for mediation of this type of tumor immunity requires the recognition of tumor antigens processed and presented by la‐positive adherent cells.

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives. III. Eradication of disseminated murine chronic leukemia cells by utilizing MDP hapten-reactive helper T-cell activity.

SummaryA previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.

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

SummaryIn 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 γ-interferon (IFNγ). Taken together, these results indicate that MAF generated by tumor-immune Lyt-1+2− T cell subset is closely related to IFNγ.

The generation of tumor-specific in vivo protective immunity in the tumor mass from mice rendered tolerant to tumor antigens

SummaryC3H/He mice were inoculated i.v. with 106 heavily X-irradiated syngeneic X5563 plasmacytoma cells 3 times at 4 day intervals. When these mice received an appropriate immunization procedure consisting of i. d. inoculation of viable tumor cells plus the surgical resection of the tumor which enables i.v. nonpresensitized mice to produce anti-X5563 immunity, they failed to develop tumor-specific immunity. This was demonstrated by the abrogation in potential of spleen and lymph node cells to generate in vivo protective immunity. In contrast, the tumor mass from X5563 tumor-bearing mice which had received the i.v. presensitization contained comparable anti-X5563 tumor neutralizing activity to that obtained from the tumor mass from nonpresensitized, X5563 tumor-bearing mice. Such an in vivo protective immunity was revealed to be mediated by tumor-specific T cells. These results demonstrate the differential generation and antitumor capability of tumor infiltrating T cells and T cells in lymphoid organs from mice which are in the tumor-specific tolerant state. The results are discussed in the context of potential utilization of tumor infiltrating in vivo protective T cells to enhance the local tumor-specific immunity in tumor-specific tolerant mice.

Cellular and Molecular Mechanisms Involved in Tumor Eradication in Vivo

Despite ample evidence of the role of T lymphocytes in rejecting tumor cells as well as allografts,(1) there are large gaps in our understanding of the effector mechanisms responsible for graft rejection. It is clearly important for both scientific and clinical reasons to define the effector mechanisms that take place within a rejecting tumor graft. The identification of an effector mechanism within a tumor graft does not necessarily imply a functional role for the mechanism in tumor destruction in vivo. Therefore, in considering the relevance of a particular mechanism, we must know whether the effector mechanism detected is subsidiary, necessary, or sufficient for tumor rejection in vivo.