Tieli Li

The restoration of the antitumor T cell response from stress-induced suppression using a traditional Chinese herbal medicine Hochu-ekki-to (TJ-41 : Bu-Zhong-Yi-Qi-Tang)

We previously reported that restraint stress impairs the antitumor immune responses through its suppressive effect on the Th1-type cytokine production from CD4+ T cells. In this study, we investigated a potential of Hochu-ekki-to (TJ-41:Bu-Zhong-Yi-Qi-Tang) to restore stress-induced immunosuppression. The oral administration of TJ-41 was able to improve a decreased cellularity in the lymph node and spleen and to improve an inhibition of tumor-specific Th1-type cytokine production, both of which were induced by repeated restraint stress in tumor-bearing mice. The oral administration of TJ-41 also induced a partial recovery of the antitumor cytolytic activity in the stress-burdened tumor-bearing mice. More importantly, the growth of tumors in stress-burdened preimmunized mice was obviously inhibited by TJ-41, and resulted in tumor-free state in 75% of the mice. Regarding the mechanisms by which TJ-41 restored the antitumor responses in stress-burdened mice, we found that the serum levels of corticosterone and interleukin-12 were normalized by TJ-41. In addition, the expression of CD80 and CD86, which both decreased in the stress-burdened mice, was restored to the normal level by TJ-41. Taken together, our results indicate that the oral administration of TJ-41 is able to restore the antitumor T cell responses in stress-burdened tumor-bearing mice by normalizing the serum corticosterone, interleukin-12 and the expression of costimulatory molecules.

Early-appearing tumor-infiltrating natural killer cells play an important role in the nitric oxide production of tumor-associated macrophages through their interferon production.

Abstract Both natural killer (NK) cells and macrophages are thought to be the main effectors responsible for early antitumor defense. In this study, we investigated the role of tumor-infiltrating NK cells in initiating nitric oxide (NO) production by tumor-associated macrophages (TAM). The in vivo depletion of NK cells prior to the i.p. inoculation of melanoma cells resulted in a significant decrease in the NO production of the TAM prepared from the peritoneal exudate cells (PEC). Such prior NK cell depletion also decreased the ability of TAM to show any antitumor activity in vitro. The addition of NG-monomethyl-L-arginine (Me-L-Arg) to the culture partially inhibited the ability of TAM to suppress the proliferation of melanoma cells and also decreased their cytolytic activity against melanoma cells. These results suggest that the TAM exhibited both cytostatic and cytolytic activities through their NO production. In an in vivo assay, the administration of Me-L-Arg permitted the more rapid growth of i.p. inoculated melanoma cells compared with the control. On the other hand, the decreased NO production of TAM, resulting from the prior NK cell depletion, was restored by the i.p. administration of interferon γ (IFNγ). In addition, the in vivo administration of anti-IFNγ mAb into mice inoculated i.p. with melanoma cells also significantly decreased the NO production of TAM in peritoneal exudate cells. Furthermore, the tumor-infiltrating NK cells produced a considerable level of IFNγ. Overall, these results indicate that early-appearing tumor-infiltrating NK cells play an important role in the NO production of TAM through their IFNγ production.

Systemic administration of interleukin-12 can restore the anti-tumor potential of B16 melanoma-draining lymph node cells impaired at a late tumor-bearing state.

We investigated the effect of the systemic administration of interleukin (IL)‐12 on the anti‐tumor potential of tumor‐draining lymph nodes (LNs). Tumor‐draining LN cells on day 10 after s.c. inoculation of B16 melanoma showed a significant anti‐tumor effect against established pulmonary metastases after in vitro expansion, whereas those on either day 20 or 30 exhibited an impaired anti‐tumor potential. However, i.p. injections of IL‐12 (0.5 μg) on days 18, 20 and 22 significantly increased the total number of tumor‐draining LN cells on day 24 and, furthermore, restored their anti‐tumor potential after in vitro expansion. In addition, i.p. injection of IL‐12 (0.1 μg) on days 20, 22, 24, 26 and 28 significantly suppressed the growth of s.c.‐inoculated B16 melanoma and finally cured the tumors in 6 of 12 mice (50%), whereas dissection of the tumor‐draining LNs on day 18, prior to the IL‐12 treatment, decreased both the IL‐12‐induced anti‐tumor effect and the percentage of cured mice (8.3%). Cured mice acquired a specific protective immunity. Collectively, our results indicate that the systemic administration of IL‐12 can restore the immunotherapeutic potential of tumor‐draining LNs, which was impaired at the late tumor‐bearing state, and that the IL‐12‐induced systemic anti‐tumor activity is preceded by the restoration of an anti‐tumor response in tumor‐draining LNs. Int. J. Cancer 75:400–405, 1998.

Characterization of B16 melanoma-specific cytotoxic T lymphocytes

Abstract A B16 melanoma-specific CD8+ T cell line (AB1) was established from the spleen cells of C57BL/6 mice cured of B16 melanoma with interleukin (IL)-12 treatment. The AB1 line exclusively used T cell receptor Vβ11. The AB1 cells exhibited a cytolytic activity against both syngeneic B16 melanoma and allogeneic P815 mastocytoma, whereas a cold inhibition assay revealed specificity of the AB1 cells against B16 melanoma. Their lostability to kill a class I loss variant of B16 melanoma was restored by the transfection of H-2Kb gene. In addition, their interferon (IFN)-γ production was significantly suppressed by the addition of anti-H-2Kb monoclonal antibody, and RT-PCR analysis showed that the AB1 line expressed the mRNA encoding IFN-γ, but not IL-4 or IL-10. The experiment using synthetic peptides of tyrosinase-related protein-2 (TRP-2) revealed that the AB1 cells could recognize TRP-2181–188 peptide. Moreover, the AB1 cells showed an in vivo antitumor effect against established pulmonary metastases of B16 melanoma. Overall, these results indicate that the Tc1-type Vβ11+ AB1 cells exert an antitumor activity against syngeneic B16 melanoma through recognition of TRP-2181–188 peptide in an H-2Kb-restricted manner.

Antitumor vaccination effect of dendritic cells can be augmented by locally utilizing Th1-type cytokines from OK432-reactive CD4+ T cells

Abstract In order to enhance the antitumor vaccination effect of dendritic cells (DC) pulsed with class I tumor peptide, we tried to utilize the local cytokine help of CD4+ T cells reactive to a streptococcal preparation OK432. DC were prepared from murine bone marrow cells by culture with both granulocyte/macrophage-colony-stimulating factor and interleukin(IL)-4. The peritumoral injections of OK432 induced OK432-reactive CD4+ T cells in the draining lymph nodes, and their in vitro production of interferon γ was thus significantly enhanced by restimulation with OK432-pulsed DC. In addition, anti-P815 mastocytoma cytotoxic T lymphocytes were generated from the in vivo OK432-treated P815-draining lymph node cells only when the lymph node cells were restimulated in vitro with the DC pulsed with both P1A peptide and OK432. Moreover, the peritumoral injections of OK432 and the subsequent vaccination of the DC, pulsed with both OK432 and P1A peptide, significantly suppressed the growth of s.c. inoculated P815. Interestingly, a significant level of IL-12 was detected in the coculture supernatant of both OK432-pulsed DC and OK432-reactive CD4+ T cells. Collectively, our results suggest that the antitumor vaccination effect of DC pulsed with class I tumor peptide could thus be effectively augmented by locally utilizing the Th1-type cytokines from OK432-reactive CD4+ T cells.

Involvement of both donor cytotoxic T lymphocytes and host NK1.1+ T cells in the thymic atrophy of mice suffering from acute graft-versus-host disease.

It has been reported that a dramatic decrease in the number of thymocytes (thymic atrophy) in mice suffering from acute graft‐versus‐host disease (GVHD) is ascribed to glucocorticoids. In this study, we examined the possibility that cellular immune responses may thus be involved in thymic atrophy. In contrast to chronic GVHD mice, acute GVHD (C57BL/6 X DBA/2) F1 (BDF1) hybrid mice, which were injected intravenously with both spleen and lymph node cells from C57BL/6 mice, showed a dramatic decrease in the number of CD4 CD8 double‐positive thymocytes 2 or 3 weeks after the induction of GVHD. A flow cytometric analysis revealed the donor‐derived T cells with either CD4 or CD8 molecules to infiltrate the thymus of the mice undergoing acute GVHD for 10 days. In a cytolytic assay, such thymus‐containing cells exhibited a cytolytic activity specific to the host cells. In addition, anti‐H‐2d cytolytic T cells showed a high level of cytolytic activity against BDF1 (H‐2bXd) thymocytes, whereas they also showed a low level of cytolytic activity against C57BL/6 (H‐2b) thymocytes, thus suggesting that the thymus‐infiltrating donor‐derived T cells killed the host thymocytes through both anti‐H‐2d‐specific and non‐specific mechanisms. Interestingly, a flow cytometric analysis revealed both the percentage and the absolute cell number of host‐derived NK1.1+ CD3+ cells to increase in the thymus of mice suffering from acute GVHD for 10 days. In addition, they also showed the cytolytic activity against YAC‐1 cells and the mRNA expression of interleukin‐12 (IL‐12) in the thymus to be also significantly augmented on day 7 after the induction of acute GVHD. Collectively, our results indicate that the cellular immune responses such as donor cytotoxic T lymphocytes and host NK1.1+ T cells are therefore involved in the thymic atrophy of mice suffering from acute GVHD.