Adoptive cell therapy using tumor-infiltrating lymphocytes for melanoma refractory to immune-checkpoint inhibitors.

To evaluate the feasibility of adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) in Japanese patients with melanoma who failed immune-checkpoint inhibitor therapy, an open-label, single-arm, pilot study was conducted. We investigated the immunological and genetic factors of the pretreatment tumor and expanded TILs that may be associated with the clinical response. The treatment protocol comprised preparation of TIL culture, lympho-depleting non-myeloablative preconditioning with cyclophosphamide and fludarabine, TIL infusion, and intravenous administration of low-dose IL-2. Three patients of clinical subtypes mucosal, superficial spreading, and acral melanoma underwent TIL-ACT. Most severe adverse events, including fever and leukopenia, were manageable with the supportive regimen specified in the protocol, suggesting that the TIL-ACT regimen is suitable for Japanese patients with melanoma. One patient showed a short-term partial response, one relatively long-stable disease, and one experienced disease progression. Whole-exome and transcriptional sequencing of isolated tumor cells and immunohistochemical analyses before TIL-ACT revealed various immunostimulatory factors, including a high tumor mutation burden and immune cell-recruiting chemokines, as well as various immunosuppressive factors including TGF-ß, VEGF, Wnt/ß-catenin, and MAPK signaling and epithelial-to-mesenchymal transition, which might influence the efficacy of TIL-ACT. Our results imply mechanisms for the antitumor effect of and resistance to TIL-ACT. Further studies of immune-resistant mechanisms of TIL-ACT are warranted. This study is registered with the UMIN Clinical Trial Registry (UMIN 000011431).

Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans.

Chronic graft-vs-host disease (cGVHD) is a multifactorial inflammatory disease that affects patients undergoing hematopoietic stem cell transplantation. Multiple organs, including the lacrimal glands (LGs), are negatively affected by cGVHD and lose function due to the resultant fibrosis. An abnormal immune response is thought to be a major factor in the development of chronic ocular GVHD, which is currently treated primarily with immunosuppressive therapies. However, all the treatments yield unsatisfactory outcomes, and additional treatment strategies are needed. To meet this unmet medical need, we aimed to elucidate an additional pathway of chronic ocular GVHD. Our findings suggest a potential association between chronic ocular GVHD pathogenesis and stress-induced cellular senescence through the senescence-associated secretory phenotype (SASP). Senescent cells produce cytokines and chemokines, such as IL-6 and CXCL9. Indeed, senescent cell accumulation was presumably associated with cGVHD development in LGs, as evidenced by the improvement in LGs after the selective elimination of senescent cells (senolysis) with ABT-263. Results in the sclerodermatous cGVHD mouse model suggest that inhibiting the major components of the SASP, including IL-6 and CXCL9, with senolytics is a potential novel strategy for treating cGVHD-affected LGs. Taken together, our results indicate a potential association between the SASP and cGVHD development in LGs and suggest that targeted senolytic treatment may be a new therapeutic option for this disease.

Functional Role of Lacrimal Gland Fibroblasts in a Mouse Model of Chronic Graft-Versus-Host Disease.

PURPOSE: This study aimed to clarify the mechanisms and assess the characteristics of the chronic graft-versus-host disease (cGVHD) fibrosis in the lacrimal gland (LG) of mice. METHODS: Histopathology of LG tissues was examined by immunohistochemistry and electron microscopy. Cultured fibroblasts derived from the LG were analyzed by phase-contrast microscopy, immunocytochemistry, flow cytometry, proliferation assay, and invasion and migration assays. RESULTS: Cultured murine LG fibroblasts in cGVHD were spindle-shaped and relatively small, whereas those from syngeneic controls were polygon-shaped and relatively large. Flow cytometric analysis showed that the LG fibroblasts in cGVHD had elevated HSP47 levels. The LG fibroblasts in cGVHD also showed increased expression of major histocompatibility complex class II. Furthermore, the proportion of Sca-1PDGFR-α cells among the LG fibroblasts in cGVHD was considerably increased compared with controls. Cell counting kit-8 assays demonstrated that the LG fibroblasts in cGVHD were highly proliferative, and cell invasion assays indicated that they were highly invasive with high migration ability. CONCLUSIONS: LG fibroblasts in cGVHD can be aberrantly activated, thereby eliciting fibrosis by producing excessive extracellular matrix, leading to LG dysfunction in mice.

Cancer-induced immunosuppressive cascades and their reversal by molecular-targeted therapy.

Immunological status in tumor tissues varies among patients. Infiltration of memory-type CD8(+) T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8(+) T cell infiltration has not been well investigated. In general, tumor-associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune-activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.

Combining carbon ion radiotherapy and local injection of α-galactosylceramide-pulsed dendritic cells inhibits lung metastases in an in vivo murine model.

PURPOSE: Our previous report indicated that carbon ion beam irradiation upregulated membrane-associated immunogenic molecules, underlining the potential clinical application of radioimmunotherapy. The antimetastatic efficacy of local combination therapy of carbon ion radiotherapy and immunotherapy was examined by use of an in vivo murine model. METHODS AND MATERIALS: Tumors of mouse squamous cell carcinoma (NR-S1) cells inoculated in the legs of C3H/HeSlc mice were locally irradiated with a single 6-Gy dose of carbon ions (290 MeV/nucleon, 6-cm spread-out Bragg peak). Thirty-six hours after irradiation, α-galactosylceramide-pulsed dendritic cells (DCs) were injected into the leg tumor. We investigated the effects on distant lung metastases by counting the numbers of lung tumor colonies, making pathologic observations, and assessing immunohistochemistry. RESULTS: The mice with no treatment (control) presented with 168 ± 53.8 metastatic nodules in the lungs, whereas the mice that received the combination therapy of carbon ion irradiation and DCs presented with 2.6 ± 1.9 (P = 0.009) at 2 weeks after irradiation. Immunohistochemistry showed that intracellular adhesion molecule 1, which activates DCs, increased from 6 h to 36 h after irradiation in the local tumors of the carbon ion-irradiated group. The expression of S100A8 in lung tissue, a marker of the lung pre-metastatic phase, was decreased only in the group with a combination of carbon ions and DCs. CONCLUSIONS: The combination of carbon ion radiotherapy with the injection of α-galactosylceramide-pulsed DCs into the primary tumor effectively inhibited distant lung metastases.

Establishment of an improved mouse model for infantile neuroaxonal dystrophy that shows early disease onset and bears a point mutation in Pla2g6.

Calcium-independent group VIA phospholipase A(2) (iPLA(2)beta), encoded by PLA2G6, has been shown to be involved in various physiological and pathological processes, including immunity, cell death, and cell membrane homeostasis. Mutations in the PLA2G6 gene have been recently identified in patients with infantile neuroaxonal dystrophy (INAD). Subsequently, it was reported that similar neurological impairment occurs in gene-targeted mice with a null mutation of iPLA(2)beta, whose disease onset became apparent approximately 1 to 2 years after birth. Here, we report the establishment of an improved mouse model for INAD that bears a point mutation in the ankyrin repeat domain of Pla2g6 generated by N-ethyl-N-nitrosourea mutagenesis. These mutant mice developed severe motor dysfunction, including abnormal gait and poor performance in the hanging grip test, as early as 7 to 8 weeks of age, in a manner following Mendelian law. Neuropathological examination revealed widespread formation of spheroids containing tubulovesicular membranes similar to human INAD. Molecular and biochemical analysis revealed that the mutant mice expressed Pla2g6 mRNA and protein, but the mutated Pla2g6 protein had no glycerophospholipid-catalyzing enzyme activity. Because of the significantly early onset of the disease, this mouse mutant (Pla2g6-inad) could be highly useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration.

Severe loss of invariant NKT cells exhibiting anti-HTLV-1 activity in patients with HTLV-1-associated disorders.

Invariant natural killer T (iNKT) cells are unique T cells that regulate the immune response to microbes, cancers, and autoimmunity. We assessed the characteristics of iNKT cells from persons infected with human T-lymphotropic virus type 1 (HTLV-1). Whereas most infected persons remain asymptomatic carriers (ACs) throughout their lives, a small proportion, usually with high equilibrium proviral loads,develop 2 diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL). We demonstrated that the frequency of iNKT, NK, and dendritic cells in the peripheral blood of HAM/TSP and ATL patients is decreased. We also observed an inverse correlation between the iNKT cell frequency and the HTLV-1 proviral load in the peripheral blood of infected persons. Notably, in vitro stimulation of peripheral blood cells with alpha-galactosylceramide led to an increase in the iNKT cell number and a subsequent decrease in the HTLV-1-infected T-cell number in samples from ACs but not HAM/TSP or ATL patients. Our results suggest that iNKT cells contribute to the immune defense against HTLV-1, and iNKT-cell depletion plays an important role in the pathogenesis of HAM/TSP and ATL. Therefore, iNKT cell-based immunotherapy may be an effective strategy for preventing these HTLV-1-associated disorders.

Differential role of thymic stromal lymphopoietin in the induction of airway hyperreactivity and Th2 immune response in antigen-induced asthma with respect to natural killer T cell function.

Asthma is an inflammatory lung disease, in which CD1d-restricted natural killer T (NKT) cells play an important pathogenic role. Also, recent reports indicated that a cytokine, thymic stromal lymphopoietin (TSLP), is essential for the development of antigen-induced asthma. Here we examined the relationship between NKT cells and TSLP in a mouse model of asthma. NKT cells express TSLP receptor as well as IL-7 receptor alpha-chain. TSLP acts on NKT cells to preferentially increase their IL-13 production but not IFN-gamma and IL-4. In an allergen-induced asthma model, the development of airway hyperreactivity, a cardinal feature of asthma, was increased in TSLP transgenic mice, whereas this effect was not observed in TSLP transgenic mice lacking NKT cells. Interestingly, in the NKT cell-lacking TSLP transgenic mice, pulmonary eosinophilia and increase in IgE did not improve. Pulmonary lymphocytes from the NKT cell-lacking TSLP transgenic mice produced much less IL-13 upon CD3 stimulation than those from NKT cell-competent TSLP transgenic mice. These resultssuggest that, in allergen-induced asthma, TSLP acts on NKT cells to enhance airway hyperreactivity by upregulating their IL-13 production, whereas eosinophilia and IgE production are not influenced.