Naoko Imai

Tumor progression inhibits the induction of multifunctionality in adoptively transferred tumor‐specific CD8+ T cells

It is becoming increasingly evident that the multifunctionality of effector cells at the single‐cell level is an important factor to predict the quality of T‐cell response in immunological protection. The significance of the multifunctionality of T cells in anti‐tumor immunity, however, remains unclear. Here, we assessed the IFN‐γ and TNF‐α production and CD107a mobilization in adoptively transferred tumor‐antigen‐specific CD8+ T cells at the single‐cell level. Tumor growth of the murine fibrosarcoma CMS5 was found to limit the induction of multifunctionality in the transferred cells. These cells exhibited insufficient acquisition of the CD25highGITRhighCD62Llow phenotype and reduced infiltration in tumor. Depletion of Treg facilitated the induction of high multifunctionality of the transferred cells even in the hosts with progressing tumor, leading to enhanced tumor regression. The multifunctionality of the transferred cells correlated with in vivo CTL activity, and T cells with high multifunctionality harvested from hosts with successful therapy induced tumor regression when re‐transferred into the tumor‐bearing hosts. These data suggest that the appearance of multifunctional CD8+ effector T cells in vivo is a critical determinant of the success of anti‐tumor immunotherapy and Treg play an important role in the mechanism inhibiting the induction of multifunctionality in effector cells.

Chemoradiotherapy-Induced Upregulation of PD-1 Antagonizes Immunity to HPV-Related Oropharyngeal Cancer

While viral antigens in human papillomavirus (HPV)-related oropharyngeal cancer (HPVOPC) are attractive targets for immunotherapy, the effects of existing standard-of-care therapies on immune responses to HPV are poorly understood. We serially sampled blood from patients with stage III-IV oropharyngeal cancer undergoing concomitant chemoradiotherapy with or without induction chemotherapy. Circulating immunocytes including CD4(+) and CD8(+) T cells, regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSC) were profiled by flow cytometry. Antigen-specific T-cell responses were measured in response to HPV16 E6 and E7 peptide pools. The role of PD-1 signaling in treatment-related immunosuppression was functionally defined by performing HPV-specific T-cell assays in the presence of blocking antibody. While HPV-specific T-cell responses were present in 13 of 18 patients before treatment, 10 of 13 patients lost these responses within 3 months after chemoradiotherapy. Chemoradiotherapy decreased circulating T cells and markedly elevated MDSCs. PD-1 expression on CD4(+) T cells increased by nearly 2.5-fold after chemoradiotherapy, and ex vivo culture with PD-1-blocking antibody enhanced HPV-specific T-cell responses in 8 of 18 samples tested. Chemoradiotherapy suppresses circulating immune responses in patients with HPVOPC by unfavorably altering effector:suppressor immunocyte ratios and upregulating PD-1 expression on CD4(+) T cells. These data strongly support testing of PD-1-blocking agents in combination with standard-of-care chemoradiotherapy for HPVOPC.

Adoptive Transfer of MAGE-A4 T-cell Receptor Gene-Transduced Lymphocytes in Patients with Recurrent Esophageal Cancer

Purpose: Preparative lymphodepletion, the temporal ablation of the immune system, has been reported to promote persistence of transferred cells along with increased rates of tumor regression in patients treated with adoptive T-cell therapy. However, it remains unclear whether lymphodepletion is indispensable for immunotherapy with T-cell receptor (TCR) gene–engineered T cells. Experimental Design: We conducted a first-in-man clinical trial of TCR gene-transduced T-cell transfer in patients with recurrent MAGE-A4–expressing esophageal cancer. The patients were given sequential MAGE-A4 peptide vaccinations. The regimen included neither lymphocyte-depleting conditioning nor administration of IL2. Ten patients, divided into 3 dose cohorts, received T-cell transfer. Results: TCR-transduced cells were detected in the peripheral blood for 1 month at levels proportional to the dose administered, and in 5 patients they persisted for more than 5 months. The persisting cells maintained ex vivo antigen-specific tumor reactivity. Despite the long persistence of the transferred T cells, 7 patients exhibited tumor progression within 2 months after the treatment. Three patients who had minimal tumor lesions at baseline survived for more than 27 months. Conclusions: These results suggest that TCR-engineered T cells created by relatively short-duration in vitro culture of polyclonal lymphocytes in peripheral blood retained the capacity to survive in a host. The discordance between T-cell survival and tumor regression suggests that multiple mechanisms underlie the benefits of preparative lymphodepletion in adoptive T-cell therapy. Clin Cancer Res; 21(10); 2268–77. ©2015 AACR.

Glucocorticoid-induced tumor necrosis factor receptor stimulation enhances the multifunctionality of adoptively transferred tumor antigen-specific CD8 + T cells with tumor regression

We have reported for the first time the significance of effector T‐cell multifunctionality in antitumor immunity, suggesting that the appearance of multifunctional/polyfunctional tumor‐specific CD8+ T cells in vivo is a critical determinant of the success of antitumor immunotherapy, and a strategy to induce multifunctionality in effector cells is required for the successful immunotherapy of hosts with progressing tumor. Glucocorticoid‐induced tumor necrosis factor receptor (GITR) stimulation has been shown to enhance antitumor immune response. However, its functional impact on adoptively transferred T cells remains unclear. Here, we analyzed the impact of GITR stimulation in vivo on the functional profiles of adoptively transferred CD8+ T cells specific for murine fibrosarcoma CMS5. GITR stimulation was found to enhance multifunctionality (interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α production and CD107a mobilization as a degranulation marker) in transferred cells at the single‐cell level. These cells exhibited upregulated expression of CD25 in draining lymph nodes and increased infiltration in tumor. Mice that received T‐cell therapy with GITR stimulation showed reduced Foxp3+CD4+ T cells among tumor infiltrating lymphocytes and increased in vivo cytotoxic T lymphocytes (CTL) activity even with progressing tumor, resulting in enhanced tumor regression. These data strengthen the idea that effector T‐cell multifunctionality is a sensitive immune correlate for successful immunotherapy against malignancy and provide an immunological rationale for effective T‐cell therapy combined with GITR stimulation. (Cancer Sci 2009; 100: 1317–1325)

T-cell receptor gene therapy targeting melanoma-associated antigen-A4 inhibits human tumor growth in non-obese diabetic/SCID/γcnull mice

Adoptive cell therapy with lymphocytes that have been genetically engineered to express tumor‐reactive T‐cell receptors (TCR) is a promising approach for cancer immunotherapy. We have been exploring the development of TCR gene therapy targeting cancer/testis antigens, including melanoma‐associated antigen (MAGE) family antigens, that are ideal targets for adoptive T‐cell therapy. The efficacy of TCR gene therapy targeting MAGE family antigens, however, has not yet been evaluated in vivo. Here, we demonstrate the in vivo antitumor activity in immunodeficient non‐obese diabetic/SCID/γcnull (NOG) mice of human lymphocytes genetically engineered to express TCR specific for the MAGE‐A4 antigen. Polyclonal T cells derived from human peripheral blood mononuclear cells were transduced with the αβ TCR genes specific for MAGE‐A4, then adoptively transferred into NOG mice inoculated with MAGE‐A4 expressing human tumor cell lines. The transferred T cells maintained their effector function in vivo, infiltrated into tumors, and inhibited tumor growth in an antigen‐specific manner. The combination of adoptive cell therapy with antigen peptide vaccination enhanced antitumor activity, with improved multifunctionality of the transferred cells. These data suggest that TCR gene therapy with MAGE‐A4‐specific TCR is a promising strategy to treat patients with MAGE‐A4‐expressing tumors; in addition, the acquisition of multifunctionality in vivo is an important factor to predict the quality of the T‐cell response during adoptive therapy with human lymphocytes. (Cancer Sci 2012; 103: 17–25)

IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting

Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution.

Peripheral Blood Regulatory T-Cell and Type 1 Helper T-Cell Population Decrease after Hepatic Artery Embolization.

PURPOSE To evaluate changes in T-cell populations in peripheral blood after bland hepatic artery embolization (HAE). MATERIALS AND METHODS Bland HAE was performed in 12 patients to treat primary (n = 5) or metastatic (n = 7) liver tumors, using microspheres and polyvinyl alcohol (n = 8) or microspheres alone (n = 4). Patient peripheral blood samples were collected within 1 month before HAE, within 1 week after HAE (early period after HAE), and 2-8 weeks after HAE (follow-up period). Peripheral blood populations of cytotoxic T lymphocytes, CD4(+) T cells, type 1 helper T cells (Th1) and type 2 helper T cells (Th2), and regulatory T cells (Treg) were evaluated using flow cytometry. Changes in T-cell populations before and after bland HAE were compared using paired t tests. RESULTS Peripheral blood CD4(+) T-cell populations decreased significantly in the early period after HAE (44.0% ± 2.2 to 34.4% ± 3.6, P < .01) and in the follow-up period (44.0% ± 2.2 to 36.3% ± 3.0, P < .01). Among the individual CD4(+) T-cell subtypes, Treg (2.5% ± 0.3 to 1.7% ± 0.2, P < .02) and Th1 (8.1% ± 1.8 to 5.6% ± 1.6, P < .02) decreased significantly in the early period after HAE only. The presence of extrahepatic disease was associated with decreasing Treg (P < .04). CONCLUSIONS After HAE, the peripheral blood T-cell environment is changed with decreases in Treg and Th1.

Changes in peripheral blood T-cell balance after percutaneous tumor ablation

Abstract Purpose: To evaluate the changes in T-cell balance in peripheral blood following percutaneous tumor ablation. Material and methods: Patients underwent thermal ablation including radiofrequency (n = 9) and microwave ablation (n = 5), or cryoablation (n = 5). Target tumors were located in the lung (n = 7), soft tissue (n = 5), liver (n = 4), and bone (n = 3). Patient peripheral blood samples were collected before and within 14 days after ablation. Peripheral blood populations of cytotoxic T-cells (CTL), type-1 (Th1) and type-2 helper T-cells (Th2), and regulatory T-cells (Treg) were measured using flow cytometry. Changes in CTL/Treg and Th1/Th2 ratios before and after ablation therapy were compared using paired t-tests. Results: Peripheral blood CTL population (27.5 ± 2.1% to 30.2 ± 2.5%, p < .03) and CTL/Treg ratios (18.8 ± 3.7% to 21.6 ± 3.6%, p < .05) increased significantly after ablation. Although a significant increase in CTL/Treg ratios was found after heat-based ablation (18.0 ± 4.4% to 21.6 ± 4.7%, p < .02), it remained unchanged after cryoablation (21.0 ± 7.0% to 21.5 ± 4.3%, p = .92). Th1/Th2 ratio (13.7 ± 3.0% to 17.2 ± 3.5%, p = .12) remained unchanged after ablation. Conclusion: Ablation therapy alters the T-cell balance by increasing the systemic CTL/Treg, ratio. Heat-based ablation might be a more effective approach than cryoablation to enhance systemic anti-tumor immunity.

Differential detection of cytoplasmic Wilms tumor 1 expression by immunohistochemistry, western blotting and mRNA quantification

Wilms tumor 1 (WT1) is considered to be a promising target of cancer treatment because it has been reported to be frequently expressed at high levels in various malignancies. Although WT1-targeted cancer treatment has been initiated, conclusive detection methods for WT1 are not established. The present study aimed to consolidate immunohistochemistry for WT1 with statistical basis. Transfected cells with forced WT1 expression yielded specific western blot bands and nuclear immunostaining; cytoplasmic immunostaining was not specifically recognized. Immunohistochemistry, western blotting, and quantitative reverse transcriptase-polymerase chain reaction were performed in 35 human cell lines using multiple WT1 antibodies and their results were quantified. Relationships among the quantified results were statistically analyzed; the nuclear immunostaining positively correlated with western blot bands and mRNA expression levels, whereas cytoplasmic immunostaining did not. These results indicate that nuclear immunostaining reflects WT1 expression but cytoplasmic immunostaining does not. The nuclear immunostaining was barely (3/541) observed in primary cancer of esophagus, bile duct, pancreas and lung. Although the present study has some limitations, the results indicate that the cytoplasmic immunostaining does not correlate with actual WT1 expression and prompts researchers to carefully evaluate target molecule expression in treatment of cancer.

Stimulation through very late antigen-4 and -5 improves the multifunctionality and memory formation of CD8⁺ T cells.

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T‐cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH‐296, we demonstrated that stimulation via very late Ag (VLA)‐4 and VLA‐5 in human and BALB/c mouse CD8+ T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR‐transgenic mouse‐derived CD8+ T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma‐derived tumor Ag, we showed that stimulation by CH‐296 improved the ability of tumor‐specific CD8+ T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3+CD4+ Treg cells in tumors. These results suggest that stimulation via VLA‐4 and VLA‐5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.