The importance of immunoregulatory T cells has become increasingly apparent. Both CD4+CD25+ T cells and CD1d-restricted NKT cells have been reported to down-regulate tumor immunity in mouse tumor models. However, the relative roles of both T cell populations have rarely been clearly distinguished in the same tumor models. In addition, CD1d-restricted NKT cells have been reported to play a critical role not only in the down-regulation of tumor immunity but also in the promotion of the immunity. However, the explanation for these apparently opposite roles in different tumor models remains unclear. We show that in four mouse tumor models in which CD1d-restricted NKT cells play a role in suppression of tumor immunity, depletion of CD4+CD25+ T cells did not induce enhancement of immunosurveillance. Surprisingly, among the two subpopulations of CD1d-restricted NKT cells, Vα14Jα18+ (type I) and Vα14Jα18− (type II) NKT cells, type I NKT cells were not necessary for the immune suppression. These unexpected results may now resolve the paradox in the role of CD1d-restricted NKT cells in the regulation of tumor immunity, in that type II NKT cells may be sufficient for negative regulation, whereas protection has been found to be mediated by α-galactosylceramide–responsive type I NKT cells.

Negative immunoregulation is a major barrier to successful cancer immunotherapy. The NKT cell is known to be one such regulator. In this study we explored the roles of and interaction between the classical type I NKT cell and the poorly understood type II NKT cell in the regulation of tumor immunity. Selective stimulation of type II NKT cells suppressed immunosurveillance, whereas stimulation of type I NKT cells protected against tumor growth even when responses were relatively skewed toward Th2 cytokines. When both were stimulated simultaneously, type II NKT cells appeared to suppress the activation in vitro and protective effect in vivo of type I NKT cells. In the absence of type I, suppression by type II NKT cells increased, suggesting that type I cells reduce the suppressive effect of type II NKT cells. Thus, in tumor immunity type I and type II NKT cells have opposite and counteractive roles and define a new immunoregulatory axis. Alteration of the balance between the protective type I and the suppressive type II NKT cell may be exploited for therapeutic intervention in cancer.

Purpose: Transforming growth factor-β (TGF-β) is an immunosuppressive cytokine, having direct suppressive activity against conventional CD4 + and CD8 + T cells and natural killer cells, thereby inhibiting tumor immunosurveillance. Here, we investigated possible synergy between anti-TGF-β (1D11) and a peptide vaccine on induction of antitumor immunity, and the mechanisms accounting for synergistic efficacy. Experimental Design: The effect of combination treatment with a peptide vaccine and anti-TGF-β was examined in a subcutaneous TC1 tumor model, as well as the mechanisms of protection induced by this treatment.Results: Anti-TGF-β significantly and synergistically improved vaccine efficacy as measured by reduction in primary tumor growth, although anti-TGF-β alone had no impact. The number of tumor antigen-specific CTL with high functional avidity as measured by IFN-γ production and lytic activity was significantly increased in vaccinated mice by TGF-β neutralization. Although TGF-β is known to play a critical role in CD4 + Foxp3 + Treg cells, Treg depletion/suppression by an anti-CD25 monoclonal antibody (PC61) before tumor challenge did not enhance vaccine efficacy, and adding anti-TGF-β did not affect Treg numbers in lymph nodes or tumors or their function. Also, TGF-β neutralization had no effect on interleukin-17-producing T cells, which are induced by TGF-β and interleukin-6. Absence of type II NKT cells, which induce myeloid cells to produce TGF-β, was not sufficient to eliminate all sources of suppressive TGF-β. Finally, the synergistic protection induced by anti-TGF-β vaccine augmentation was mediated by CD8 + T cells since anti-CD8 treatment completely abrogated the effect. Conclusions: These results suggest that TGF-β blockade may be useful for enhancing cancer vaccine efficacy. (Clin Cancer Res 2009;15(21):6560-9)

Though TGF-b inhibition enhances antitumor immunity mediated by CD8 1 T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF-b blockade, we tested the effect of anti-TGF-b combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti-TGF-b antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti-TGF-b antibodies compared to vaccinated mice without anti-TGF-b, suggesting that anti-TGF-b synergistically enhanced irradiated tumor vaccine efficacy. CD8 1 T-cell depletion completely abrogated the vaccine efficacy, and so protection required CD8 1 T cells. Depletion of CD25 1 T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti-TGF-b did not change the number of CD25 1 T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d-restricted NKT cells, which have been reported to induce TGF-b production by MDSC through an IL-13-IL-4R-STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti-TGF-b enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF-b levels found in patients with cancer and that the effect is not dependent on TGF-b solely from CD4 1 CD25 1 T regulatory cells or the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway.The success of cancer immunotherapy depends on overcoming immune suppression in patients. There are multiple mechanisms suggested to suppress antitumor immunity. TGF-b plays important roles in several of such mechanisms of immune suppression.TGF-b is a highly pleiotropic cytokine and can be produced by many lymphoid and nonlymphoid cells. 1 TGF-b can directly enhance growth, metastasis, and angiogenesis of some tumors. [2][3][4][5][6][7] In antitumor immunity, tumor antigen-specific cytotoxic T lymphocytes (CTLs) play crucial roles in eradicating tumors. However, TGF-b inhibits the antitumor immune response at several levels including the production of perforin, granzyme A, granzyme B, FAS ligand, and IFN-c by CTLs in vitro and in vivo. 8 In human patients with melanoma, antigen-specific CD8 þ T-cell effector function in vitro is inhibited by the addition of TGF-b. 9 TGF-b also influences dendritic cells (DCs), which are critical in priming protective CD4 þ Th 1-and CD8 þ CTLmediated antitumor responses. TGF-b can inhibit DC migration and antigen transport to draining lymph nodes (LNs) within murine skin tumors, effectively obstructing T-cell activation. 10 In addition to such an immobilization of DCs, TGF-b may also decrease DC numbers by escalating apoptosis 11 and limit their func...

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Although Japanese traditional herbal medicine (Kampo) has been widely applied to the treatment of various diseases, including cancer, their mechanisms of action have not yet been elucidated in detail, particularly regarding their role in tumor immunology. The present study investigated the antitumor effects of the Japanese Kampo medicine, ninjin'yoeito (NYT; Ren-Shen-Yang-Rong-Tang in Chinese), which was orally administered with or without an irradiated tumor cell vaccine to a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine in a prophylactic setting significantly delayed tumor growth in mice fed a control diet, whereas a diet containing NYT alone did not exert any antitumor effects in vivo. However, the inhibition of tumor growth was significantly greater in vaccinated mice fed the NYT diet compared with in vaccinated mice given the control diet. These results suggest that NYT synergistically enhances the effects of the antitumor vaccine. The depletion of cluster of differentiation (CD)8+ T cells abrogated these effects, indicating that antitumor activity required CD8+ T cells. Furthermore, reductions in CD4+ CD25+ and forkhead box protein 3+ T regulatory cell numbers were more apparent between vaccinated mice fed the NYT diet and non-vaccinated mice fed the control diet than between vaccinated mice and non-vaccinated mice fed the control diet, suggesting that the weaker impact of T regulatory cells contributes to the augmentation of antitumor immunity by CD8+ T cells in vaccinated mice fed with NYT. Overall, these results indicate that NYT synergistically enhances the effects of the prophylactic tumor vaccine mediated by CD8+ T cells and that this Japanese Kampo medicine has potential as a useful adjuvant agent for cancer immunotherapy.

Although the Japanese traditional herbal medicine (Kampo), Juzentaihoto (JTT), has been reported to have antitumor effects in several tumor models, its role in tumor immunology remains controversial. In the present study, we tested whether oral administration of JTT enhances antitumor immunity in CD1d−/− mice, in which immunosuppression was partially relieved due to the lack of NKT cells. In a subcutaneous murine syngeneic CT26 colorectal tumor model, JTT had no impact on tumor growth in wild type (WT) BALB/c mice. However, the growth rate of tumors was significantly slower in CD1d−/− mice than in WT mice. Surprisingly, JTT significantly delayed tumor growth in such CD1d−/− mice. In vivo depletion of CD8+ T cells revealed that CD8+ T cells are required for JTT’s antitumor activity. Moreover, tumor-reactive cytotoxic T-lymphocytes were detected exclusively in JTT-treated mice with well-controlled tumors. JTT did not affect the number of tumor-infiltrating CD4+ regulatory T cells. On the contrary, JTT increased the degranulation marker CD107a+ CD8+ T cells and decreased Ly6G+ Ly6Clo polymorphonuclear myeloid-derived suppressor cells in tumor-infiltrating lymphocytes, most probably contributing to the suppression of tumor growth in JTT-treated mice. Nonetheless, JTT had no impact on the proportion of monocytic myeloid-derived suppressor cells. In conclusion, our results indicate that in the absence of NKT cells, JTT augments antitumor immunity by CD8+ T cells, suggesting that this Kampo medicine is a promising anticancer adjuvant when negative immune regulation is partially relieved.

To test the efficacy of chemical disinfectants against bacterial biofilms in hemodialysis equipment, a Center for Disease Control and Prevention (CDC)-Biofilm Reactor was used to create biofilms. Methylobacterium radiotolerance was isolated from the hemodialysis fluid and used as the test organism. We examined the efficacy of sodium hypochlorite (NaOCl) in elimination of planktonic cells compared to that in the case of biofilms. Planktonic bacteria were completely eliminated at 50 parts per million (ppm) of NaOCl, which is the lowest concentration for clinical use. The viable cell count in the biofilm reached its minimum value around a logarithmic reduction value (LRV) of 6, when the concentration was raised to 1000 ppm and the reaction time was extended by 1 hour or more. Furthermore, at 200 ppm, the LRV was elevated depending on the time. And the LRV while maintaining static conditions for 6 hours at 200 ppm was similar to that of short time at 1000 ppm. These results suggest that NaOCl has sufficient bactericidal activity even for biofilms at a practical concentration and reaction time, and that the CDC-Biofilm Reactor is an effective tool for finding useful disinfection conditions.