Yoriyuki Kamiryo

Soluble branched (1,4)-β-D-glucans from Acetobacter species enhance antitumor activities against MHC class I-negative and -positive malignant melanoma through augmented NK activity and cytotoxic T-cell response

We previously found that an extracellular polysaccharide, AC‐1, produced by Acetobacter polysaccharogenes composed of (1,4)‐β‐D‐glucan with branches of glucosyl residues showed a strong activity to induce production of interleukin (IL)‐12 p40 and tumor necrosis factor‐α by macrophage cell lines in vitro via Toll‐like receptor‐4 signaling. In the present study, we examined the effects of oral administration of AC‐1 on protection against 2 types of murine B16 melanoma lines, major histocompatibility complex (MHC) class I‐negative B16L and MHC class I gene‐transfected B16Kb cells. Mice were inoculated subcutaneously with B16L or B16Kb cells on day 0 and administrated intragastrically with AC‐1 or PBS once every 5 days from 1 day before tumor inoculation. The tumor growth was severely retarded in AC‐1‐treated mice after subcutaneous inoculation with B16L or B16Kb cells. The AC‐1‐treated mice showed augmented natural killer (NK) cell activity against B16L cells, and in vivo depletion of NK cells by antiasialoGM1 antibody (Ab) treatment abrogated the antitumor activity in AC‐1‐treated mice. On the other hand, AC‐1‐treated mice inoculated with B16Kb cells developed a significantly higher level of cytotoxic T‐lymphocyte response against B16Kb cells, and in vivo depletion of CD8+ T cells by anti‐CD8 mAb treatment abrogated the antitumor activity. Thus, AC‐1 augmented antitumor activity against different tumors via augmentation of different antitumor mechanisms. These results suggest a possible prophylactic application of AC‐1 for human neoplasms irrespective of expression levels of their MHC class I molecules.

Donor CD4 T Cells Are Critical in Allogeneic Stem Cell Transplantation against Murine Solid Tumor

Nonmyeloablative allogeneic stem cell transplantation (SCT) has been used for various malignancies, although detailed mechanisms of antitumor effects remain unclear. We showed that a nonmyeloablative allogeneic SCT regimen, which consists of mixed chimerism induced by an injection of donor spleen and bone marrow cells followed by cyclophosphamide treatment and a donor lymphocyte infusion (DLI), exerted antitumor effects on established murine bladder tumor, MBT-2. An expansion of donor CD4 T cells accompanied by transient but vigorous IFN-gamma production was detected shortly after DLI. In vivo neutralization of IFN-gamma or depletion of CD4 T cells from DLI abolished the antitumor effects, indicating an indispensable role of donor CD4 T cells producing IFN-gamma. Donor as well as host CD8 T cells accumulated in the tumor region with time. Importantly, depletion of CD8 T cells from DLI did not reverse the suppression of tumor growth, indicating that CD4 T cells play a more essential role in mediating early antitumor effects. Furthermore, tumor-specific response of host CD8 T cells was suggested. These results not only provide the first evidence of nonmyeloablative allogeneic SCT for the treatment of bladder tumor but also elucidate detailed mechanisms of antitumor effects provoked by DLI.

Renal Cancer Treatment with Low Levels of Mixed Chimerism Induced by Nonmyeloablative Regimen Using Cyclophosphamide in Mice

Recently, much attention has been paid to nonmyeloablative allogeneic stem cell transplantation for the treatment of metastatic renal cancer. Mature donor T cells cause graft-versus-host disease (GVHD) although they are also the main mediators of the beneficial graft-versus-tumor activity associated with this treatment. Hence, the segregation of the graft-versus-tumor activity from GVHD is an important challenge in managing the clinical course of treatment. We previously reported a series of studies regarding the allograft tolerance induced by allogeneic spleen cells (with bone marrow cells) and cyclophosphamide in mice. Here, we show a modified cyclophosphamide-induced tolerance system for the treatment of murine renal cell carcinoma, RENCA, by shifting the equal balance between graft-versus-host and host-versus-graft reactions toward graft-versus-host reaction with donor lymphocyte infusion. Our results clearly show the antitumor activity against RENCA with only low levels of mixed chimerism in the periphery and the in vivo and in vitro acquired immunity against RENCA even when mixed chimerism is almost undetectable. Because the withdrawal of mixed chimerism reduces the risk of GVHD, the antitumor activity is thus sequentially segregated from the initial GVHD in our model. We believe that this is the first unique model system of nonmyeloablative allogeneic hemopoietic cell transplantation to ever be reported for the treatment of renal cancer.

Posttransplant administration of cyclophosphamide and donor lymphocyte infusion induces potent antitumor immunity to solid tumor

Purpose: Nonmyeloablative allogeneic stem cell transplantation (SCT) has been increasingly used for the treatment of hematologic and solid malignancies, and mature donor T cells are considered to be the main effectors of the graft-versus-tumor (GVT) activity. However, the association between degree of donor chimerism and intensity of GVT effects has not been fully elucidated. We recently proposed a unique nonmyeloablative cell therapy using posttransplant cyclophosphamide and donor lymphocyte infusion, by which a significant antitumor effect against murine renal cell carcinoma, RENCA, was induced, although the level of mixed chimerism was relatively low. In this study, we attempted to clarify a role of chimerism for in vivo antitumor effects on GVT effects in radiation-associated nonmyeloablative SCT. Experimental Design: We assessed antitumor effects on RENCA tumors and the degree of donor chimerism after several doses of irradiation followed by allogeneic SCT and compared the results with those of cyclophosphamide-based cell therapy. Results: Allogeneic SCT following sublethal irradiation (6 Gy) induced almost complete donor chimerism, whereas cyclophosphamide-based cell therapy produced low levels of donor chimerism. Nonetheless, GVT activity was much more potent in cyclophosphamide-based cell therapy than irradiation-conditioned SCT. Furthermore, cyclophosphamide-conditioned SCT induced more potent immune reconstitution with less severe graft-versus-host disease than irradiation-conditioned SCT. Conclusions: Our results indicate that a high level of chimerism is not essential for the in vivo antitumor effect of nonmyeloablative allogeneic cell therapy against solid tumor and that the recovery of peripheral lymphocytes after the initial immunosuppression might be a critical event for the elicitation of in vivo antitumor effects of that treatment modality.

Cyclophosphamide-Using Nonmyeloablative Allogeneic Cell Therapy against Renal Cancer with a Reduced Risk of Graft-versus-Host Disease

Purpose: Much attention has been paid to nonmyeloablative allogeneic stem cell transplantation for the treatment of renal cancer. We recently proposed a cyclophosphamide-using nonmyeloablative cell therapy in which donor lymphocyte infusion (DLI) was carried out after the tolerance induction to donor cells. In considering the clinical application of the cyclophosphamide-using cell therapy, attempts to reduce graft-versus-host disease (GVHD) are crucial. The aim of the present study was to modify the cyclophosphamide-using cell therapy to reduce the risk of GVHD while preserving the antitumor activity against renal cancer. Experimental Design: We assessed whether a delay in performing DLI from day 1 to day 5 after the cyclophosphamide treatment could reduce the risk of GVHD while preserving antitumor activity against RENCA, a murine carcinogen-induced renal cell carcinoma, in the cyclophosphamide-using cell therapy. Results: Regarding the in vivo antitumor effect, there was no difference between DLI on day 1 and day 5 after the cyclophosphamide treatment, whereas the histologic findings of the small intestine showed that the cyclophosphamide-using cell therapy with DLI on day 5 decreased the risk of GVHD. In addition, the acquired immunity against RENCA was also observed in the RENCA-rejected mice that had been treated with DLI on day 5. Conclusions: Our results show that a delay in DLI during cyclophosphamide-using nonmyeloablative cell therapy can dissociate graft-versus-tumor effects from GVHD by reducing the risk of GVHD.

Antitumor activity of recombinant Bacille Calmette-Guérin secreting interleukin-15-Ag85B fusion protein against bladder cancer

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is used for the treatment of bladder cancer. The recruitment of neutrophlis to the bladder after BCG instillation exerts anti-tumor activity against bladder tumor. We have recently demonstrated that interleukin (IL)-17A produced by γδ T cells played a role in the recruitment of neutrophlis to the bladder after BCG instillation. IL-15 is known to play an important role in neutrophil migration during inflammation. We previously constructed a recombinant BCG strain expressing the fusion protein of IL-15 and Ag85B (BCG-IL-15) for prevention of Mycobacterium tuberculosis infection. Here we compared the efficacy of the BCG-IL-15 in protection against bladder cancer with that of rBCG-Ag85B (BCG). Six-week-old female C57BL/6 mice were inoculated with MB49 bladder tumor cells in the bladder and subsequently intravesically inoculated with BCG or BCG-IL-15. BCG-IL-15 treatment significantly prolonged survival of mice inoculated with bladder cancer cells compared with BCG treatment. Infiltration of neutrophils was significantly elevated in BCGB-IL-15 treated mice accompanied by increased chemokines (MIP-2 and MIP-1α) in the bladder. Thus, BCG-IL-15 exerted additive effect on Infiltration of neutrophils in the bladder. BCG-IL-15 may be a promising drug for non-muscle invasive bladder cancer.

Allogeneic cell therapy from immunized donors with tumor antigen peptide enhances the antitumor effect after cyclophosphamide-using non-myeloablative allogeneic hematopoietic cell transplantation.

Non‐myeloablative allogeneic stem cell transplantation is an option for the treatment of hematological malignancies as well as solid tumors. We recently proposed a cyclophosphamide‐using non‐myeloablative cell therapy in which donor lymphocytes infusion (DLI) was carried out after tolerance induction to donor cells. In this study, we tested the possibility that the cyclophosphamide‐using cell therapy could be augmented by pre‐immunization of donors before DLI. We initially assessed whether or not the cyclophosphamide‐using cell therapy could also show antitumor effect against subcutaneously established colon 26 carcinoma. As a tumor antigen‐derived peptide for colon 26, we used AH1, an immunodominant H‐2Ld‐binding peptide derived from the envelope protein (gp70) of an endogenous murine leukemia virus. The cyclophosphamide‐using cell therapy with the DLI from donors which were pre‐immunized with the AH1 peptide was compared with that from non‐immunized mice. The cyclophosphamide‐using cell therapy significantly suppressed subcutaneously established colon 26 carcinoma, and the tumor‐rejected mice acquired the tumor‐specific protective immunity. When combined with the DLI from donors that were immunized with AH1, antitumor effect of the cyclophosphamide‐using cell therapy was significantly augmented. The DLI from tumor peptide‐immunized donors showed no influence on donor chimerism and bodyweight of the treated mice, indicating no increased risk of graft‐versus‐host disease. Tumor‐specific cytotoxic T lymphocytes could be generated from tumor‐rejected mice. Our results indicate that the cyclophosphamide‐using non‐myeloablative cell therapy with the DLI from tumor peptide‐immunized donors is a useful protocol to augment graft‐versus‐tumor effect without exacerbation of graft‐versus‐host disease. (Cancer Sci 2009; 100: 138–143)