Satoru Miura

Both Regulatory T Cells and Antitumor Effector T Cells Are Primed in the Same Draining Lymph Nodes during Tumor Progression

The peripheral tolerance mechanism prevents effective antitumor immunity, even though tumor cells possess recognizable tumor-associated Ags. Recently, it has been elucidated that regulatory T cells (Treg) play a critical role in maintaining not only self-tolerance, but also tolerance of tumor cells. However, because the Treg that maintain self-tolerance arise naturally in the thymus and are thought to be anergic in peripheral, it is still unclear where and when Treg for tumor cells are generated. In this study we analyze tumor-draining lymph nodes (LNs) and demonstrate that both antitumor effector T cells and Treg capable of abrogating the antitumor reactivity of the effector T cells are primed in the same LNs during tumor progression. The regulatory activity generated in tumor-draining LNs exclusively belonged to the CD4+ T cell subpopulation that expresses both CD25 and a high level of CD62L. Forkhead/winged helix transcription factor gene expression was detected only in the CD62LhighCD4+CD25+ T cells. CD62LhighCD4+CD25+ Treg and CD62LlowCD4+CD25+ T cells, which possess effector T cell functions, had comparable expression of LFA-1, VLA-4, CTLA-4, lymphocyte activation gene-3, and glucocorticoid-induced TNFR. Thus, only CD62L expression could distinguish regulatory CD4+CD25+ cells from effector CD4+CD25+ cells in draining LNs as a surface marker. The Treg generated in tumor-draining LNs possess the same functional properties as the Treg that arise naturally in the thymus but recognize tumor-associated Ag. CD62LhighCD4+CD25+ Treg contained a subpopulation that expressed CD86. Blocking experiments revealed that ligation of CTLA-4 on effector T cells by CD86 on Treg plays a pivotal role in regulating CD4+ effector T cells.

Reciprocal CD4+ T-Cell Balance of Effector CD62Llow CD4+ and CD62LhighCD25+ CD4+ Regulatory T Cells in Small Cell Lung Cancer Reflects Disease Stage

Purpose: Small cell lung cancer (SCLC) possesses high tendency to disseminate. However, SCLC patients with paraneoplastic syndrome mediated by immunity against onconeural antigens remain in limited-stage disease (LD) without distant metastases. Cumulative evidence regulates that a balance between immune and regulatory T (Treg) cells determines the magnitude of immune responses to not only self-antigens but also tumor-associated antigens. The purpose of this study was to elucidate the immunologic balance induced in SCLC patients. Experimental Design: We analyzed T cells in the peripheral blood of 35 consecutive SCLC patients, 8 long-term survivors, and 19 healthy volunteers. Results: Purified CD4+ T cells with down-regulated expression of CD62L (CD62Llow) produced IFN-γ, interleukin (IL)-4, and IL-17, thus considered to be immune effector T cells (Teff). Significantly more Teff cell numbers were detected in LD-SCLC patients than that of extended-stage SCLC (ED-SCLC). By contrast, induction of CD62LhighCD25+ CD4+ Treg cells was significantly higher in ED-SCLC patients. Long-term survivors of SCLC maintained a high Teff to Treg cell ratio, whereas patients with recurrent disease exhibited a low Teff to Treg cell ratio. Teff cells in LD-SCLC patients included more IL-17–producing CD4+ T cells (Th17). Moreover, dendritic cells derived from CD14+ cells of LD-SCLC patients secreted more IL-23. Conclusion: These results show that CD4+ T-cell balance may be a biomarker that distinguishes ED-SCLC from LD-SCLC and predicts recurrence. This study also suggests the importance of inducing Teff cells, particularly Th17 cells, while eliminating Treg cells to control systemic dissemination of SCLC.

DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked is an immunogenic target of cancer stem cells

Abstract Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, survives through potentially lethal stresses such as chemotherapy and radiation treatment. Since the survival of this subpopulation of cancer stem cells (CSC) plays a critical role in recurrence, it must be eradicated in order to cure cancer. We previously reported that vaccination with CD133+ murine melanoma cells exhibiting biological CSC features induced CSC-specific effector T cells. These were capable of eradicating CD133+ tumor cells in vivo, thereby curing the parental tumor. In the current study, we indicated that DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133+ tumor cells. Vaccination with DDX3X primed specific T cells, resulting in protective and therapeutic antitumor immunity. The DDX3X-primed CD4+ T cells produced CD133+ tumor-specific IFNγ and IL-17 and mediated potent antitumor therapeutic efficacy. DDX3X is expressed in various human cancer cells, including lung, colon, and breast cancer cells. These results suggest that anti-DDX3X immunotherapy is a promising treatment option in efforts to eradicate CSC in the clinical setting.

Generation of Anti-tumour Effector T Cells from Naïve T Cells by Stimulation with Dendritic/tumour Fusion Cells

Tumour‐draining lymph node T cells are an excellent source of effector T cells that can be used in adoptive tumour immunotherapy because they have already been sensitized to tumour‐associated antigens in vivo. However, such tumour‐specific immune cells are not readily obtained from the host due to poor immunogenicity of tumours and reduced host immune responses. One obstacle in implementation of adoptive immunotherapy has been insufficient sensitization and expansion of tumour‐specific effector cells. In this study, we aim to improve adoptive immunotherapy by generating anti‐tumour effector T cells from naïve T lymphocytes. We attempted to achieve this by harnessing the advantages of dendritic cell (DC)‐based anti‐cancer vaccine strategies. Electrofusion was routinely employed to produce fusion cells with 30–40% efficiency by using the poorly immunogenic murine B16/F10 cell line, D5 cells, and DC generated from bone marrow cells. CD62L‐positive T cells from spleens of naïve mice and the fusion cells were cocultured with a low concentration of IL‐2. After 9 days of culture, the antigen‐specific T cells were identified with an upregulation of CD25 and CD69 expression and a downregulation of CD62L expression. These cells secreted IFN‐γ upon stimulation with irradiated tumour cells. Moreover, when transferred into mice with 3‐day established pulmonary metastases, these cells with coadministration of IL‐2 exhibited anti‐tumour efficacy. In contrast, naïve T cells cocultured with a mixture of unfused DC and irradiated tumour cells did not exhibit anti‐tumour efficacy. Our strategy provides the basis for a new approach in adoptive T cell immunotherapy for cancer.

Expression of thymidylate synthase, orotate phosphoribosyltransferase and dihydropyrimidine dehydrogenase in thymic epithelial tumors

BACKGROUND It remains unclear whether thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) expressions are associated with the pathogenesis of thymic epithelial tumors. Therefore, we investigated the expression of TS, OPRT and DPD in thymic epithelial tumors. PATIENTS AND METHODS Fifty-six patients with thymic epithelial tumors were included in this study. Tumors sections were stained by immunohistochemistry for TS, OPRT, DPD, microvessel density (MVD) determined by CD34, and p53. We also conducted in vitro study of TS, OPRT and DPD expression using thymic carcinoma, thymic tumor and thymic fibroblast cell lines. RESULTS TS, OPRT and DPD were expressed in 61%, 48% and 41%, respectively. High grade malignancy is significantly associated with higher expression of TS, OPRT and DPD in thymic epithelial tumors. These biomarkers were closely associated with p53 and MVD, and the overexpression of TS and DPD was a prognostic marker for predicting poor outcome in univariate analysis. Our in vitro study showed that marked overexpression of TS and OPRT was observed in thymic carcinoma cells, but not in thymic tumor cells, or thymic fibroblast cells. CONCLUSIONS The expression of TS, OPRT and DPD was closely related to the grade of malignancy in thymic epithelial tumors. A positive expression of TS, DPD and OPRT might be an important factor in predicting the effectiveness of 5-FU based chemotherapy in this disease.

Expression of excision repair cross-complementation group 1, breast cancer susceptibility 1, and β III-tubulin in thymic epithelial tumors.

Background: We investigated the clinical significance of excision repair cross-complementation group 1 (ERCC1), breast cancer susceptibility 1 (BRCA1), and class III &bgr;-tubulin (TUBB3) expression in thymic epithelial tumors. Method: Fifty-six patients with thymic epithelial tumors were included in this study. Tumors sections were stained by immunohistochemistry for ERCC1, BRCA1, TUBB3, microvessel density, and p53. Results: ERCC1, BRCA1, and TUBB3 were expressed in 48%, 50%, and 27%, respectively. The expression of ERCC1, BRCA1, and TUBB3 was significantly correlated with the grade of malignancy in thymic epithelial tumors. These biomarkers were closely associated with p53 and microvessel density and were a prognostic marker for predicting poor outcome. We also found that overexpression of ERCC1 and TUBB3 was associated with resistance to platinum- and taxane-based chemotherapy. Conclusion: An expression of ERCC1, BRCA1, and TUBB3 was correlated strongly with each other and was significantly associated with a poor outcome in thymic epithelial tumors. High-ERCC1 and TUBB3 expressions might be associated with resistance to platinum- and taxane-based chemotherapy, respectively.

Critical Roles of Chemoresistant Effector and Regulatory T Cells in Antitumor Immunity after Lymphodepleting Chemotherapy.

Antitumor immunity is augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects in lymphopenic recipients. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8+ T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents; however, the percentage of CD4+CD25+ Foxp3+ Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemoresistant Tregs following CPA treatment and transfer of naive CD4+ T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8+ T cells were responsible for this augmentation. Using Rag2−/− mice or depletion of recipient CD8+ T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4+ T cells enhanced the proliferation of CD8+ T cells and the priming of tumor-specific CD8+ T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies.

DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations

The specific mechanisms how lung cancer cells harboring epidermal growth factor receptor (EGFR) activating mutations can survive treatment with EGFR-tyrosine kinase inhibitors (TKIs) until they eventually acquire treatment-resistance genetic mutations are unclear. The phenotypic diversity of cancer cells caused by genetic or epigenetic alterations (intratumor heterogeneity) confers treatment failure and may foster tumor evolution through Darwinian selection. Recently, we found DDX3X as the protein that was preferentially expressed in murine melanoma with cancer stem cell (CSC)-like phenotypes by proteome analysis. In this study, we transfected PC9, human lung cancer cells harboring EGFR exon19 deletion, with cDNA encoding DDX3X and found that DDX3X, an ATP-dependent RNA helicase, induced CSC-like phenotypes and the epithelial-mesenchymal transition (EMT) accompanied with loss of sensitivity to EGFR-TKI. DDX3X expression was associated with upregulation of Sox2 and increase of cancer cells exhibiting CSC-like phenotypes, such as anchorage-independent proliferation, strong expression of CD44, and aldehyde dehydrogenase (ALDH). The EMT with switching from E-cadherin to N-cadherin was also facilitated by DDX3X. Either ligand-independent or ligand-induced EGFR phosphorylation was inhibited in lung cancer cells that strongly expressed DDX3X. Lack of EGFR signal addiction resulted in resistance to EGFR-TKI. Moreover, we found a small nonadherent subpopulation that strongly expressed DDX3X accompanied by the same stem cell-like properties and the EMT in parental PC9 cells. The unique subpopulation lacked EGFR signaling and was highly resistant to EGFR-TKI. In conclusion, our data indicate that DDX3X may play a critical role for inducing phenotypic diversity, and that treatment targeting DDX3X may overcome primary resistance to EGFR-TKI resulting from intratumor heterogeneity.

A case of severe thrombocytopaenia associated with acute HIV-1 infection

A 23-year-old man was admitted to our hospital with severe thrombocytopaenia. He had unprotected sexual contact 6 weeks earlier. He was diagnosed with acute HIV infection by means of HIV RNA viral load testing and HIV-associated thrombocytopaenia. Although his thrombocytopaenia improved immediately with short-term dexamethasone therapy, this effect was not sustained after cessation of therapy. Antiretroviral therapy including raltegravir was initiated, and the patient recovered from severe thrombocytopaenia within several days. The findings from this case suggest that acute HIV infection should be suspected with unexplained thrombocytopaenia, and that antiretroviral therapy is the treatment of choice for severe HIV-associated thrombocytopaenia, even when in the early period following acquisition of the virus.

Recurrent Interstitial Lung Disease Induced By Various Therapies forNon-Small Cell Lung Cancer

Erlotinib is a human epidermal growth factor receptor type 1 tyrosine kinase inhibitor which is used for non-small cell lung cancer treatment. Interstitial lung disease has been reported as an adverse event of erlotinib. We report the case of a 39-year-old man with erlotinib-induced interstitial lung disease in a non-small cell lung cancer patient. Although interstitial lung disease had improved by steroid therapy, palliative radiotherapy recalled the pneumonitis beyond the radiation fields. After the pneumonitis was well controlled, the patient was started on irinotecan, but the interstitial lung disease recurred shortly thereafter. We may have to abandon further cytotoxic therapies to avoid the recurrence of interstitial lung disease in patients who develop erlotinib-induced interstitial lung disease once.