CAR-Modified Vγ9Vδ2 T Cells Propagated Using a Novel Bisphosphonate Prodrug for Allogeneic Adoptive Immunotherapy.

The benefits of CAR-T therapy could be expanded to the treatment of solid tumors through the use of derived autologous αß T cell, but clinical trials of CAR-T therapy for patients with solid tumors have so far been disappointing. CAR-T therapy also faces hurdles due to the time and cost intensive preparation of CAR-T cell products derived from patients as such CAR-T cells are often poor in quality and low in quantity. These inadequacies may be mitigated through the use of third-party donor derived CAR-T cell products which have a potent anti-tumor function but a constrained GVHD property. Vγ9Vδ2 TCR have been shown to exhibit potent antitumor activity but not alloreactivity. Therefore, in this study, CAR-T cells were prepared from Vγ9Vδ2 T (CAR-γδ T) cells which were expanded by using a novel prodrug PTA. CAR-γδ T cells suppressed tumor growth in an antigen specific manner but only during a limited time window. Provision of GITR co-stimulation enhanced anti-tumor function of CAR-γδ T cells. Our present results indicate that, while further optimization of CAR-γδ T cells is necessary, the present results demonstrate that Vγ9Vδ2 T cells are potential source of 'off-the-shelf' CAR-T cell products for successful allogeneic adoptive immunotherapy.

Acceptable surface limits (ASLs) of skin sensitizers derived from the local lymph node assay (LLNA): BrdU-ELISA EC1.6 values and their relationships to known sensitization potency information.

Skin sensitization is an extremely important risk factor for occupational health and safety, and it would be desirable to set health-based exposure limits (HBELs) for the quantitative risk assessment (QRA) based on the skin sensitizing potencies of chemical. We attempted to set acceptable surface limits (ASLs) as HBELs for skin sensitizers in the workplace based on the local lymph node assay (LLNA): BrdU-ELISA EC1.6 values. To calculate the ASLs, a safety assessment factor (SAF)interspecies value of 6, based on the EC1.6 values/human repeat insult patch test (HRIPT) NOEL ratios, a SAFinterindividual value of 10, and a SAFfrequency/duration value of 3 were applied, referring to previous literatures on SAFs for skin sensitization QRA, and the composite SAF was calculated as 180. The ASLs (mg/100 cm2 ) derived thus for 33 chemicals ranged from 0.001 to 10.417. Comparison of the ranges with known human sensitization potency classes and GHS subcategories revealed that use of GHS Category 1A chemicals needs to be controlled to ensure surface residue levels of less than 1 mg/100 cm2 . To minimize sensitization risks, a quantitative sensitization risk assessment method for chemicals and appropriate risk management are necessary. This report provides a potentially useful ASL-based method of managing sensitization risk derived from LLNA: BrdU-ELISA EC1.6 values, comparison of the ASLs and known human sensitization potency data showed that GHS subcategorization results would be a primary information notifying ASL ranges to be required for minimizing the sensitization risk.

Identification of genomic locus responsible for experimentally induced testicular teratoma 1 (ett1) on mouse Chr 18.

Spontaneous testicular teratomas (STTs) composed by various kinds of tissues are derived from primordial germ cells (PGCs) in the fetal testes of the mouse. In contrast, intra-testicular grafts of the mouse strain (129/Sv-Ter (+/+)) fetal testes possessed the ability to develop the experimental testicular teratomas (ETTs), indistinguishable from the STTs at a morphological level. In this study, linkage analysis was performed for exploration of possible candidate genes involving in ETT development using F2 intercross fetuses derived from [LTXBJ × 129/Sv-Ter (+/+)] F1 hybrids. Linkage analysis with selected simple sequence length polymorphisms along chromosomes 18 and 19, which have been expected to contain ETT-susceptibility loci, demonstrated that a novel recessive candidate gene responsible for ETT development is located in 1.1 Mb region between the SSLP markers D18Mit81 and D18Mit184 on chromosome 18 in the 129/Sv-Ter (+/+) genetic background. Since this locus is different from the previously known loci (including Ter, pgct1, and Tgct1) for STT development, we named this novel gene "experimental testicular teratoma 1 (ett1)". To resolve the location of ett1 independently from other susceptibility loci, ett1 loci was introduced in a congenic strain in which the distal segment of chromosome 18 in LTXBJ strain mice had been replaced by a 1.99 Mbp genomic segment of the 129/Sv-Ter (+/+) mice. Congenic males homozygous for the ett1 loci were confirmed to have the ability to form ETTs, indicating that this locus contain the gene responsible for ETTs. We listed candidate genes included in this region, and discussed about their possible involvement in induction of ETTs.

Comparison of the acceptable surface limits derived from the skin sensitization potency for workers and swab residue limits determined from the permitted daily exposure derived from the systemic effects.

Allergic contact dermatitis is a common occupational and environmental health problem and setting of health-based exposure limits (HBELs) to prevent induction of skin sensitization is strongly desired. When manufacturing pharmaceuticals in a shared facility, cleaning validation using surface residue levels (SRLs) derived from permitted daily exposures (PDEs) is conducted to avoid cross-contamination from the perspective of protecting patients; however, it is unclear whether the SRLs are sufficient to prevent induction of skin sensitization for workers as well. In this study, we compared acceptable surface limits (ASLs) derived from acceptable exposure levels (AELs) based on EC1.6 obtained from local lymph node assay (LLNA): BrdU-ELISA for occupational risk management of skin sensitizers with PDE-based SRLs. ASLs for 1,4-phenylenediamine (GHS skin sensitization sub-category 1A), isoeugenol (sub-category 1A), and methyl methacrylate (sub-category 1B) were compared with SRLs based on the PDEs derived from their systemic effects. The results yielded an SRL for 1,4-phenylenediamine (PDE: 0.8 mg/day) of 30 mg/100 cm2, almost 1,000 times higher than ASL (0.031 mg/100 cm2) derived from its skin sensitization potency. SRL for isoeugenol (PDE: 3.1 mg/day) was 130 mg/100 cm2, over 500 times higher than ASL (0.18 mg/100 cm2). For methyl methacrylate (PDE: 5 mg/day) as well, SRL (200 mg/100 cm2) was higher, but it was within 20 times the ASL (10 mg/100 cm2). These results showed that ASL-based risk management is extremely important especially for strong sensitizers classified as GHS sub-category 1A for occupational skin sensitization risk management.

Antigen delivery targeted to tumor-associated macrophages overcomes tumor immune resistance.

Immune checkpoint inhibitors and adoptive transfer of gene-engineered T cells have emerged as novel therapeutic modalities for hard-to-treat solid tumors; however, many patients are refractory to these immunotherapies, and the mechanisms underlying tumor immune resistance have not been fully elucidated. By comparing the tumor microenvironment of checkpoint inhibition-sensitive and -resistant murine solid tumors, we observed that the resistant tumors had low immunogenicity. We identified antigen presentation by CD11b+F4/80+ tumor-associated macrophages (TAMs) as a key factor correlated with immune resistance. In the resistant tumors, TAMs remained inactive and did not exert antigen-presenting activity. Targeted delivery of a long peptide antigen to TAMs by using a nano-sized hydrogel (nanogel) in the presence of a TLR agonist activated TAMs, induced their antigen-presenting activity, and thereby transformed the resistant tumors into tumors sensitive to adaptive immune responses such as adoptive transfer of tumor-specific T cell receptor-engineered T cells. These results indicate that the status and function of TAMs have a significant impact on tumor immune sensitivity and that manipulation of TAM functions would be an effective approach for improving the efficacy of immunotherapies.

Applicability of an Integrated Testing Strategy consisting of in silico, in chemico and in vitro assays for evaluating the skin sensitization potencies of isocyanates.

The skin sensitization potential of chemicals has been traditionally assessed using regulatory accepted in vivo methods, such as guinea pig maximization test or mouse local lymph node assays (LLNAs). A huge effort to reduce and replace the use of animals for safety assessments of chemicals because of regulatory requirements and ethical issues is presently underway, and alternative non-animal methods have been greatly developed. So far, a few studies have investigated the sensitization potencies of isocyanates which is a group of highly reactive chemicals that are known to be occupational allergens. The present study evaluated nine commonly used isocyanates using an in vivo LLNA and assessed the applicability of an Integrated Testing Strategy (ITS) consisting of an in silico Derek Nexus prediction, an in chemico direct peptide reactivity assay (DPRA), and an in vitro human Cell Line Activation Test (h-CLAT) to isocyanates. All nine isocyanates were evaluated as positive using the LLNA, Derek Nexus and DPRA, whereas seven chemicals tested positive using the h-CLAT: hexamethylene diisocyanate tested negative, and 1,5-diisocyanatonaphthalene could not be examined because of a solubility issue. When assessed using the ITS, the positive/negative evaluations of skin sensitization hazard were consistent with those assessed using the LLNA for all nine chemicals. However, the potency prediction results of the ITS tended to be underestimated, compared with those of the LLNA. The data presented in this work provide insights into the performance of non-animal testing approaches for evaluating the skin sensitization potencies of isocyanates.

Signal-transducing adaptor protein-2 promotes generation of functional long-term memory CD8+ T cells by preventing terminal effector differentiation.

Long-surviving memory CD8+ T cells generated by stimulation with appropriate tumor-associated antigens are the most aggressive and persistent tumoricidal effectors. In this event of memory CD8+ T cell development, the signal transducer and activator of transcription (STAT) proteins function as the crucial intracellular signaling molecules, but the regulatory mechanism of STATs in CD8+ T cells is not fully understood. In this study, we report for the first time, by using murine vaccination models, that signal-transducing adaptor protein-2 (STAP2) maintains the cytotoxicity of long-lived memory CD8+ T cells by controlling a STAT3/suppressor of cytokine signaling 3 (SOCS3) cascade. Following T cell activation, STAP2 expression was transiently reduced but was subsequently recovered and augmented. Analysis using small-interfering RNA (siRNA) demonstrated that restored STAP2 expression was associated with the activation of STAT3/SOCS3 signals and maintenance of cytotoxic T lymphocytes (CTLs) secondary responses by preventing their differentiation into terminal effector cells. Notably, this STAP2-dependent memory differentiation was observed in the spleen, but not in the lymph nodes (LNs). These findings indicate an essential role for STAP2 in the generation of a high-quality memory CD8+ CTLs periphery, and suggest the therapeutic potential of STAP2 in cancer patients.

Nanogel-based immunologically stealth vaccine targets macrophages in the medulla of lymph node and induces potent antitumor immunity.

Because existing therapeutic cancer vaccines provide only a limited clinical benefit, a different vaccination strategy is necessary to improve vaccine efficacy. We developed a nanoparticulate cancer vaccine by encapsulating a synthetic long peptide antigen within an immunologically inert nanoparticulate hydrogel (nanogel) of cholesteryl pullulan (CHP). After subcutaneous injection to mice, the nanogel-based vaccine was efficiently transported to the draining lymph node, and was preferentially engulfed by medullary macrophages but was not sensed by other macrophages and dendritic cells (so-called "immunologically stealth mode"). Although the function of medullary macrophages in T cell immunity has been unexplored so far, these macrophages effectively cross-primed the vaccine-specific CD8(+) T cells in the presence of a Toll-like receptor (TLR) agonist as an adjuvant. The nanogel-based vaccine significantly inhibited in vivo tumor growth in the prophylactic and therapeutic settings, compared to another vaccine formulation using a conventional delivery system, incomplete Freund's adjuvant. We also revealed that lymph node macrophages were highly responsive to TLR stimulation, which may underlie the potency of the macrophage-oriented, nanogel-based vaccine. These results indicate that targeting medullary macrophages using the immunologically stealth nanoparticulate delivery system is an effective vaccine strategy.