Akihiro Hosoi

Zoledronate facilitates large-scale ex vivo expansion of functional γδ T cells from cancer patients for use in adoptive immunotherapy

BACKGROUND Human gammadelta T cells can be activated by phospho-antigens and aminobisphosphonates such as zoledronate. Because they can kill tumor cells in a major histocompatibility complex (MHC)-unrestricted manner, adoptive transfer of activated gammadelta T cells may represent a novel cancer immunotherapy. We tested whether gammadelta T cells from advanced cancer patients can be expanded by zoledronate. METHODS Peripheral blood mononuclear cells from healthy donors and patients with advanced non-small cell lung cancer, bone metastatic breast or prostate cancer, or lung metastatic colorectal cancer, were stimulated with zoledronate (5 microM) and interleukin (IL)-2 (1000 IU/mL) for 14 days. The phenotype and function of the expanded gammadelta T-cell populations from healthy donors and cancer patients were compared. RESULTS Gammadelta T cells from cancer patients and healthy donors responded to zoledronate equally well in terms of both phenotype and function. gammadelta T cells grew rapidly in vitro and expression of effector molecules, such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, perforin, granzyme B, FasL and TRAIL, increased over time. Cytotoxicity peaked on days 12-14, and proliferation continued up to 14 days, during which time>1x10(9) gammadelta T cells could be obtained from a starting sample of 45-70 mL peripheral blood. DISCUSSION Using the agent zoledronate, already widely used in the clinic, we have established that efficient large-scale ex vivo expansion of gammadelta T cells from cancer patients is possible. These cells exert potent cytotoxicity and may be used for autologous cellular immunotherapy of cancer.

Adoptive cytotoxic T lymphocyte therapy triggers a counter‐regulatory immunosuppressive mechanism via recruitment of myeloid‐derived suppressor cells

Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T‐cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel‐1‐specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor‐bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN‐γ, exerted anti‐tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b+Gr1+ myeloid‐derived suppressor cells (MDSCs). Notably, CD11b+Gr1intLy6G−Ly6C+ monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen‐specific CD8+ T cells. The anti‐tumor activity of the adoptively‐transferred CTLs and the accumulation of MDSCs both depended on IFN‐γ production on recognition of tumor antigens by the former. In CCR2−/− mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter‐regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment‐induced recruitment of these MDSCs would improve the efficacy of immunotherapy.

Cytotoxic T Lymphocytes Block Tumor Growth Both by Lytic Activity and IFNγ-Dependent Cell-Cycle Arrest

Matsushita and colleagues used microarray gene expression analysis coupled with fluorescent ubiquitination-based cell-cycle indicator (fucci) analysis and flow cytometry of a B16 murine model of melanoma to demonstrate that the predominant mechanism of CTL therapy in regulating tumor growth is via IFNγ-mediated cell-cycle arrest. To understand global effector mechanisms of CTL therapy, we performed microarray gene expression analysis in a murine model using pmel-1 T-cell receptor (TCR) transgenic T cells as effectors and B16 melanoma cells as targets. In addition to upregulation of genes related to antigen presentation and the MHC class I pathway, and cytotoxic effector molecules, cell-cycle–promoting genes were downregulated in the tumor on days 3 and 5 after CTL transfer. To investigate the impact of CTL therapy on the cell cycle of tumor cells in situ, we generated B16 cells expressing a fluorescent ubiquitination-based cell-cycle indicator (B16-fucci) and performed CTL therapy in mice bearing B16-fucci tumors. Three days after CTL transfer, we observed diffuse infiltration of CTLs into the tumor with a large number of tumor cells arrested at the G1 phase of the cell cycle, and the presence of spotty apoptotic or necrotic areas. Thus, tumor growth suppression was largely dependent on G1 cell-cycle arrest rather than killing by CTLs. Neutralizing antibody to IFNγ prevented both tumor growth inhibition and G1 arrest. The mechanism of G1 arrest involved the downregulation of S-phase kinase-associated protein 2 (Skp2) and the accumulation of its target cyclin-dependent kinase inhibitor p27 in the B16-fucci tumor cells. Because tumor-infiltrating CTLs are far fewer in number than the tumor cells, we propose that CTLs predominantly regulate tumor growth via IFNγ-mediated profound cytostatic effects rather than via cytotoxicity. This dominance of G1 arrest over other mechanisms may be widespread but not universal because IFNγ sensitivity varied among tumors. Cancer Immunol Res; 3(1); 26–36. ©2014 AACR. See related commentary by Riddell, p. 23

Impact of culture medium on the expansion of T cells for immunotherapy

BACKGROUND AIMS Encouraging evidence of clinical benefits from cancer immunotherapy is beginning to accumulate in several clinical trials. Cancer immunotherapy is based on two main methods, active vaccination and cell-transfer therapy. The ex vivo expansion of T cells is required to monitor vaccine-induced antigen-specific T cells or prepare large numbers of reactive lymphocytes for adoptive transfer. METHODS We examined the influence of culture medium on T-cell growth, cytotoxicity and phenotype after activation using immobilized anti-CD3 monoclonal antibody or Zoledronate stimulation. Peripheral blood mononuclear cells (PBMC) were cultured in RPMI, AIM-V or OpTmizer with or without autologous serum. RESULTS When supplemented with sufficient serum, RPMI was a good culture medium for T-cell expansion following anti-CD3 stimulation. Addition of autologous serum to AIM-V or OpTmizer increased the numbers of cells obtained to a similar extent, but their phenotype and function were quite different. Activated T cells cultured with OpTmizer mediated greater cytotoxicity than any other culture. Regardless of the media used, the main population expanded after CD3 stimulation was CD3(+) CD8(+). While more CD3(+) CD4(+) T cells were induced in RPMI and AIM-V, more CD3(-) CD56(+) cells and CD3(+) CD56(+) T cells were induced in OpTmizer. When cells were stimulated by Zoledronate for 14 days, approximately 7.2 times and 11.5 times more gammadelta T cells were obtained in OpTmizer than AIM-V or RPMI, respectively. CONCLUSIONS Successful immunotherapy depends on the selection of appropriate culture media to support efficient expansion of the type of T cell desired.

Increased susceptibility to spontaneous lung cancer in mice lacking LIM-domain only 7

LIM‐domain only (LMO) 7 is a multifunctional protein that is predicted to regulate the actin cytoskeleton, assembly of adherens junctions in epithelial cells, and gene expression. LMO7 was highly expressed in the mouse lung and predominantly localized to the apical membrane domain of bronchiolar epithelial cells. Although mice lacking LMO7 were viable and fertile in specific pathogen‐free conditions, they developed protruding epithelial lesions in the terminal and respiratory bronchioles and alveolar ducts at 14–15 weeks of age. Furthermore, they tended to develop spontaneous adenocarcinoma in the lung at over 90 weeks of age. The cumulative incidence ratios of lung cancer were 22% in LMO7−/– mice and 13% in LMO7+/– mice whereas no primary lung cancer was observed in wild‐type mice. Ex vivo analyses of the cancer cells showed numerical chromosome abnormalities and tumorigenicity in nude mice. These results suggest that LMO7 can act as a tumor suppressor whose deficiency confers a genetic predisposition to naturally occurring lung cancer. (Cancer Sci 2009; 100: 608–616)

An Immunogram for the Cancer-Immunity Cycle: Towards Personalized Immunotherapy of Lung Cancer.

Introduction: The interaction of immune cells and cancer cells shapes the immunosuppressive tumor microenvironment. For successful cancer immunotherapy, comprehensive knowledge of antitumor immunity as a dynamic spatiotemporal process is required for each individual patient. To this end, we developed an immunogram for the cancer‐immunity cycle by using next‐generation sequencing. Methods: Whole exome sequencing and RNA sequencing were performed in 20 patients with NSCLC (12 with adenocarcinoma, seven with squamous cell carcinoma, and one with large cell neuroendocrine carcinoma). Mutated neoantigens and cancer germline antigens expressed in the tumor were assessed for predicted binding to patients’ human leukocyte antigen molecules. The expression of genes related to cancer immunity was assessed and normalized to construct a radar chart composed of eight axes reflecting seven steps in the cancer‐immunity cycle. Results: Three immunogram patterns were observed in patients with lung cancer: T‐cell–rich, T‐cell–poor, and intermediate. The T‐cell–rich pattern was characterized by gene signatures of abundant T cells, regulatory T cells, myeloid‐derived suppressor cells, checkpoint molecules, and immune‐inhibitory molecules in the tumor, suggesting the presence of antitumor immunity dampened by an immunosuppressive microenvironment. The T‐cell–poor phenotype reflected lack of antitumor immunity, inadequate dendritic cell activation, and insufficient antigen presentation in the tumor. Immunograms for both the patients with adenocarcinoma and the patients with nonadenocarcinoma tumors included both T‐cell–rich and T‐cell–poor phenotypes, suggesting that histologic type does not necessarily reflect the cancer immunity status of the tumor. Conclusions: The patient‐specific landscape of the tumor microenvironment can be appreciated by using immunograms as integrated biomarkers, which may thus become a valuable resource for optimal personalized immunotherapy.

Memory Th1 Cells Augment Tumor-Specific CTL following Transcutaneous Peptide Immunization

Targeting dendritic cells in vivo by transcutaneous peptide immunization (TCI) represents an efficient immunization strategy to induce tumor-specific CTL because it reflects the physiologic conditions occurring during pathogen infection. Here we show that including a Th1 peptide in TCI can activate preexisting memory Th1 (mTh1) responses and thereby enhance the CTL response. For this purpose, peptide-25, a major Th1 epitope of Ag85B from Mycobacterium tuberculosis, was selected. We adoptively transferred peptide-25-specific mTh1 cells and hgp100-specific naive CTL (pmel-1 TCR transgenic) into C57BL/6 mice. Subsequently, mice were transcutaneously immunized with CTL peptide (hgp100) and Th1 peptide (peptide-25). Five days after TCI, the frequency and function of pmel-1 cells was monitored by intracellular IFN-gamma staining, ELISPOT, and in vivo cytotoxicity assays. TCI efficiently expanded hgp100-specific, IFN-gamma-producing, strongly cytotoxic CD8(+) T cells. Concurrent activation of mTh1 cells by peptide-25 induced a 1.5-fold increase in the number of hgp100-specific CTL with enhanced effector functions. Furthermore, TCI elicited not only prophylactic but also therapeutic antitumor responses that were augmented by peptide-25. These results show that TCI facilitates peptide-specific activation of CD4(+) T cells, responsible for the augmenting effect of peptide-25 on the hgp100-specific CTL response. Because a significant proportion of the Japanese population has been vaccinated with Bacillus Calmette-Guerin, they are likely to possess Ag85B- or peptide-25-specific mTh1 cells. Therefore, concomitant activation of Ag85B- or peptide-25-specific mTh1 cells together with tumor-specific CTL by TCI might augment antitumor immune responses in a sizeable fraction of patients.

Targeting spatiotemporal expression of CD137 on tumor-infiltrating cytotoxic T lymphocytes as a novel strategy for agonistic antibody therapy.

CD137 (4-1BB) is an important costimulatory ligand and a potent stimulator of T-cell responses. It has been used therapeutically to stimulate immunity against several solid malignancies as well as to modulate susceptibility to autoimmune disease and infection. However, clinical trials of anti-CD137 agonistic antibody have been suspended because of deleterious side effects. To overcome this problem, we fine-tuned the combination of adoptive transfer of tumor-specific cytotoxic T lymphocytes (CTL) and anti-CD137 monoclonal antibody (mAb) treatment. B16 melanoma cells (1×106) were implanted subcutaneously in C57BL/6 mice. On day 9, CTLs (1×107) were intravenously injected into tumor-bearing mice. Transferred CTL distributed throughout the body and infiltrated into the tumor. CD137 expression was upregulated on tumor-infiltrating CTL, but not in other tissues or other cell types. Therefore, mice received anti-CD137 mAb (100 &mgr;g) 3 days after CTL transfer. interferon-&ggr; was produced in the tumor only by transferred CTL, not recipient-derived cells. The functional CTLs in the tumor were increased and interferon-&ggr; production per cell was augmented by anti-CD137 treatment. It was not detected in CTL found in other tissues. Consistent with this, no organ damage was observed on anti-CD137 treatment. On the basis of the spatiotemporal expression of CD137 on tumor-infiltrating CTLs, anti-CD137 mAb selectively activated these tumor-infiltrating cells, augmented their antitumor activity and greatly decreased tumor growth. Tumor-specific CTL can guide agonistic anti-CD137 mAb to the tumor and in turn, become functionally augmented. Thus, CD137 mAb therapy may become feasible again in combination with tumor-specific CTL therapy.

Identification of Individual Cancer-Specific Somatic Mutations for Neoantigen-Based Immunotherapy of Lung Cancer

Introduction: Two strategies for selecting neoantigens as targets for non–small cell lung cancer vaccines were compared: (1) an “off‐the‐shelf” approach starting with shared mutations extracted from global databases and (2) a personalized pipeline using whole‐exome sequencing data on each patients tumor. Methods: The Catalogue of Somatic Mutations in Cancer database was used to create a list of shared missense mutations occurring in more than 1% of patients. These mutations were then assessed for predicted binding affinity to HLA alleles of 15 lung cancer patients, and potential neoantigens (pNeoAgs) for each patient were selected on this basis. In the personalized approach, pNeoAgs were selected from missense mutations detected by whole‐exome sequencing of the patients own samples. Results: The list of shared mutations included 22 missense mutations for adenocarcinoma and 18 for squamous cell carcinoma (SCC), resulting in a median of 10 off‐the‐shelf pNeoAgs for each adenocarcinoma (range 5–13) and 9 (range 5–12) for each SCC. In contrast, a median of 59 missense mutations were identified by whole‐exome sequencing (range 33–899) in adenocarcinoma and 164.5 (range 26–232) in SCC. This resulted in a median of 46 pNeoAgs (range 13–659) for adenocarcinoma and 95.5 (range 10–145) for SCC in the personalized set. We found that only one or two off‐the‐shelf pNeoAgs were included in the set of personalized pNeoAgs—and then in only three patients, with no overlap seen in the remaining 12 patients. Conclusions: Use of an off‐the‐shelf pipeline is feasible but may not be satisfactory for most patients with non–small cell lung cancer. We recommend identifying personal mutations by comprehensive genome sequencing for developing neoantigen‐targeted cancer immunotherapies.

Dendritic cell vaccine with mRNA targeted to the proteasome by polyubiquitination.

Dendritic cells (DCs) transfected with mRNA encoding tumor-associated antigens (TAAs) can induce tumor-specific T-cell responses. To potentiate this, we transfected mature DCs (mDCs) with mRNA encoding TAA targeted to the proteasome. DCs were generated from bone marrow cells by culture with 20 ng/ml GM-CSF and maturation with 1 microg/ml LPS. These mDCs were then electroporated with 10 microg of mRNA. Antigen presentation after electroporation with in vitro transcribed mRNA was compared with mRNA from a construct of the TAA preceded by ubiquitin. Proteasomal targeting of mRNA encoding cotranslationally ubiquitinated antigen was found to enhance intracellular degradation of target protein, and result in more efficient priming and expansion of TAA-specific CD8(+) T-cells. We therefore suggest that RNA-transfected DC vaccine efficacy could be improved by the use of mRNA targeted to the proteasome.