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Abstract Tumor-specific CD8+ T cells play a pivotal role in antitumor immunity and are a key target of immunotherapeutic approaches. Intratumoral CD8+ T cells are heterogeneous; Tcf1+ stemlike CD8+ T cells give rise to their cytotoxic progeny—Tim-3+ terminally differentiated CD8+ T cells. However, where and how this differentiation process occurs has not been elucidated. We herein show that terminally differentiated CD8+ T cells can be generated within tumor-draining lymph nodes (TDLN) and that CD69 expression on tumor-specific CD8+ T cells controls its differentiation process through regulating the expression of the transcription factor TOX. In TDLNs, CD69 deficiency diminished TOX expression in tumor-specific CD8+ T cells, and consequently promoted generation of functional terminally differentiated CD8+ T cells. Anti-CD69 administration promoted the generation of terminally differentiated CD8+ T cells, and the combined use of anti-CD69 and anti–programmed cell death protein 1 (PD-1) showed an efficient antitumor effect. Thus, CD69 is an attractive target for cancer immunotherapy that synergizes with immune checkpoint blockade.

Abstract Cancer immunotherapy utilizes our immune system to attack cancer cells and is an extremely promising strategy for cancer treatment. Although immune-checkpoint blockade, such as anti-PD-1 (programmed cell death 1) antibody, has demonstrated significant enhancement of anti-tumor immunity and has induced notable clinical outcomes, its response rates remain low, and adverse effects are always a matter of concern; therefore, new targets for cancer immunotherapy are always desired. In this situation, new concepts are needed to fuel the investigation of new target molecules for cancer immunotherapy. We propose that CD69 is one such target molecule. CD69 is known to be an activation marker of leukocytes and is also considered a crucial regulator of various immune responses through its interacting proteins. CD69 promotes T-cell retention in lymphoid tissues via sphingosine-1-phosphate receptor 1 (S1P1) internalization and also plays roles in the pathogenesis of inflammatory disorders through interacting with its functional ligands Myl9/12 (myosin light chains 9, 12a and 12b). In anti-tumor immunity, CD69 is known to be expressed on T cells in the tumor microenvironment (TME) and tumor-draining lymph nodes (TDLNs). We revealed that CD69 negatively regulates the effector function of intratumoral T cells and importantly controls the ‘exhaustion’ of CD8 T cells. In addition, we and others showed that either CD69 deficiency or the administration of anti-CD69 monoclonal antibody enhances anti-tumor immunity. Thus, CD69 is an attractive target for cancer immunotherapy.

While IFNγ is a well-known cytokine that actively promotes the type I immune response, it is also known to suppress the type II response by inhibiting the differentiation and proliferation of Th2 cells. However, the mechanism by which IFNγ suppresses Th2 cell proliferation is still not fully understood. We found that IFNγ decreases the expression of growth factor independent-1 transcriptional repressor (GFI1) in Th2 cells, resulting in the inhibition of Th2 cell proliferation. The deletion of theGfi1gene in Th2 cells results in the failure of their proliferation, accompanied by an impaired cell cycle progression. In contrast, the enforced expression of GFI1 restores the defective Th2 cell proliferation, even in the presence of IFNγ. These results demonstrate that GFI1 is a key molecule in the IFNγ-mediated inhibition of Th2 cell proliferation.

Abstract Glioblastoma is the most common and aggressive type of brain tumor with high recurrence and fatality rates. Although various therapeutic strategies have been explored, there is currently no effective treatment for glioblastoma. Recently, the number of immunotherapeutic strategies has been tested for malignant brain tumors. Invariant natural killer T (iNKT) cells play an important role in anti-tumor immunity. To address if iNKT cells can target glioblastoma to exert anti-tumor activity, we assessed the expression of CD1d, an antigen-presenting molecule for iNKT cells, on glioblastoma cells. Glioblastoma cells from 10 of 15 patients expressed CD1d, and CD1d-positive glioblastoma cells pulsed with glycolipid ligand induced iNKT cell-mediated cytotoxicity in vitro. Although CD1d expression was low on glioblastoma stem-like cells, retinoic acid, which is the most common differentiating agent, upregulated CD1d expression in these cells and induced iNKT cell-mediated cytotoxicity. Moreover, intracranial administration of human iNKT cells induced tumor regression of CD1d-positive glioblastoma in orthotopic xenografts in NOD/Shi-scid IL-2RγKO (NOG) mice. Thus, CD1d expression represents a novel target for NKT cell-based immunotherapy for glioblastoma patients.

MicroRNAs (miRNAs), one of small non-coding RNAs, regulate many cell functions through their post-transcriptionally downregulation of target genes. Accumulated studies have revealed that miRNAs are involved in hematopoiesis. In the present study, we investigated effects of miR-669m overexpression on hematopoiesis in mouse in vivo, and found that erythroid differentiation was inhibited by the overexpression. Our bioinformatic analyses showed that candidate targets of miR-669m which are involved in the erythropoiesis inhibition are A-kinase anchoring protein 7 (Akap7) and X-linked Kx blood group (Xk) genes. These two genes were predicted as targets of miR-669m by two different in silico methods and were upregulated in late erythroblasts in a public RNA-seq data, which was confirmed with qPCR. Further, miR-669m suppressed luciferase reporters for 3' untranslated regions of Akap7 and Xk genes, which supports these genes are direct targets of miR-669m. Physiologically, miR-669m was not expressed in the erythroblast. In conclusion, using miR-669m, we found Akap7 and Xk, which may be involved in erythroid differentiation, implying that manipulating these genes could be a therapeutic way for diseases associated with erythropoiesis dysfunction.

Background: Invariant natural killer T (iNKT) cells are known as CD1d-restricted T cells that express the invariant T-cell receptors (TCR) Vα24 and Vβ11 in humans and specifically recognize glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d-positive tumor cells presenting glycolipid antigens and indirect cytotoxicity by activating other cytotoxic immune cells or regulating CD1d-positive immunosuppressive cells in the tumor microenvironment. Although we previously reported that αGalCer-activated NKT cells exert a potent perforin-dependent cytotoxic activity against a wide variety of human tumor cell lines, the direct recognition of CD1d-negative tumors is controversial and the mechanism is unknown. Here we clarify whether iNKT cells recognize and exhibit cytotoxicity toward leukemia cells in a CD1d-independent manner and identify the molecule that recognizes CD1d-negative leukemia cells. Methods: Purified iNKT cells were generated from peripheral blood mononuclear cells (PBMCs) of healthy adult volunteer donors. PBMCs were cultured in complete RPMI 1640 medium for 9-14 days in the presence of 100 U/mL of recombinant human IL-2 and 200 ng/mL of αGalCer. The iNKT cells were then isolated with an autoMACS Pro separator using FITC-labeled anti-Vα24 antibody (clone, C15) and anti-FITC microbeads. We evaluated the cytotoxic activity of iNKT cells toward CD1d-negative leukemia cells within four days after isolation using a CD107a assay for degranulation, cytometric bead array for cytokine production, and cytotoxicity assay in vitro and in vivo. For in vivo cytotoxicity assays, NOG mice were inoculated with 1 × 106 K562-luc cells on day 0 and with 4 × 106 human iNKT cells on day 1. Gene knock-out (KO) was performed using a CRISPR/Cas9 system. T-cell or NK receptor-KO iNKT cells were used for experiments three or four days after electroporation of the Cas9 protein and guide RNA CRISPR ribonucleoprotein complex. Patient-derived leukemia cells were obtained from PBMCs or bone marrow mononuclear cells of pre-treatment pediatric patients. All studies were approved by the institutional review board and the Animal Care and Use Committee of Chiba University. Results: We observed that iNKT cells degranulated and released Th1 cytokines when co-cultured with CD1d-negative leukemia cells (K562, HL-60, REH, and CD1d-KO U937) as well as αGalCer-loaded CD1d-positive leukemia cells (Jurkat), and showed in vitro cytotoxicity toward these CD1d-negative leukemia cells. This CD1d-independent degranulation decreased over time after isolation and was not restored with re-stimulation by αGalCer. The cytotoxicity of iNKT cells toward K562 cells was confirmed in vivo by comparsion with survival curves of K562-inoculated NOG mice given iNKT cells or PBS alone (log-rank, p= 0.016). To identify the receptors contributing to the CD1d-independent recognition and cytotoxicity against CD1d-negative leukemia cells, we first focused on costimulatory receptors, which are also known as activating NK receptors and are expressed on iNKT cells such as NKG2D, DNAM-1, 2B4, LFA-1, and CD2, and analyzed cytotoxicity after blocking these receptors with antibodies. We found that all costimulatory receptors that we assessed contributed to cytotoxicity toward CD1d-negative leukemia cells. Next, we analyzed cytotoxicity of TCR-KO iNKT cells toward CD1d-negative leukemia cells to confirm the contribution of TCR to CD1d-independent recognition. Notably, TCR-KO iNKT cells showed decreased degranulation, Th1 cytokine release, and cytotoxicity toward K562 cells more so than iNKT cells with KO of NK receptors such as LFA-1(CD11a) or CD2. To assess the clinical application potential of adoptive iNKT cell immunotherapy for leukemia treatment, we analyzed degranulation of iNKT cells using patient-derived leukemia cells. We found iNKT cells degranulation using cells from four out of five myeloid leukemia cases, but only one out of eight BCP-ALL cases (p = 0.032). Conclusion: Primary iNKT cells activated by αGalCer can recognize and show anti-tumor effects toward leukemia cells in an unrestricted manner via CD1d. The TCR also has an important role in recognizing CD1d-negative leukemia cells and multiple NK receptors assist in cytotoxicity. Adoptive iNKT cell immunotherapy may be effective in treating myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Glioblastoma is one of the most aggressive forms of cancers and has a poor prognosis. Genomewide analyses have revealed that a set of core signaling pathways, the p53, RB, and RTK pathways, are commonly deregulated in glioblastomas. However, the molecular mechanisms underlying the tumorigenicity of glioblastoma are not fully understood. Here, we show that the lysine deacetylase SIRT2 is required for the proliferation and tumorigenicity of glioblastoma cells, including glioblastoma stem cells. Furthermore, we demonstrate that SIRT2 regulates p73 transcriptional activity by deacetylation of its C-terminal lysine residues. Our results suggest that SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells and that SIRT2 may be a promising molecular target for the therapy of glioblastoma.

Mycobacterium bovis Bacille Calmette-Guérin (BCG) has been shown to possess potent anti-tumor activity particularly in various animal models, while the cellular and molecular mechanisms underlying its activity are not well understood. We found that lipomannan (BCG-LM), a lipophilic component of the mycobacterial cell envelope, specifically inhibits tumor growth and induces the infiltration of eosinophils at local tumor invasion sites. In contrast, neither lipoarabinomannan (BCG-LAM) nor the cell wall of Mycobacterium bovis BCG (BCG-CW) exerted anti-tumor immunity. BCG-LM enhances cytotoxic activity of eosinophils via the increased production of superoxide. Global transcriptomic analyses of BCG-LM-pulsed dendritic cells identified C-C motif ligand (CCL) 5 as a crucial chemokine for the anti-tumor immunity induced by BCG-LM, indicating that CCL5 plays an important role for the accumulation of eosinophils in the tumor microenvironment. Furthermore, BCG-LM and memory Th2 cells exerted a synergetic effect on tumor progression by cooperatively enhancing the eosinophil function. Thus, this study revealed an un-identified BCG-LM-mediated anti-tumor mechanism via superoxide produced by infiltrated eosinophils in the tumor microenvironment. Since BCG-LM activates this unique pathway, it may have potent therapeutic potential as immune cell therapy for cancer patients.

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours(1-3). APC is involved in the proteasome-mediated degradation of beta-catenin, through its interaction with beta-catenin, GSK-3beta and Axin(4,5). APC also interacts with the microtubule cytoskeleton(6-8) and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells'. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends(10). However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. We show here that APC interacts with the kinesin superfamily (KIF) 3A-KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and beta-catenin are transported along microtubules by KAP3-KIF3A-KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration.

The adenomatous polyposis coli gene ( APC ) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. Here the APC gene product is shown to bind through its armadillo repeat domain to a Rac-specific guanine nucleotide exchange factor (GEF), termed Asef. Endogenous APC colocalized with Asef in mouse colon epithelial cells and neuronal cells. Furthermore, APC enhanced the GEF activity of Asef and stimulated Asef-mediated cell flattening, membrane ruffling, and lamellipodia formation in MDCK cells. These results suggest that the APC-Asef complex may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function.