CD69 Imposes Tumor-Specific CD8+ T-cell Fate in Tumor-Draining Lymph Nodes.

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.

The pathogenetic significance of exhausted T cells in a mouse model of mature B cell neoplasms.

Dysfunctional anti-tumor immunity has been implicated in the pathogenesis of mature B cell neoplasms, such as multiple myeloma and B cell lymphoma; however, the impact of exhausted T cells on disease development remains unclear. Therefore, the present study investigated the features and pathogenetic significance of exhausted T cells using a mouse model of de novo mature B cell neoplasms, which is likely to show immune escape similar to human patients. The results revealed a significant increase in PD-1+ Tim-3- and PD-1+ Tim-3+ T cells in sick mice. Furthermore, PD-1+ Tim-3+ T cells exhibited direct cytotoxicity with a short lifespan, showing transcriptional similarities to terminally exhausted T cells. On the other hand, PD-1+ Tim-3- T cells not only exhibited immunological responsiveness but also retained stem-like transcriptional features, suggesting that they play a role in the long-term maintenance of anti-tumor immunity. In PD-1+ Tim-3- and PD-1+ Tim-3+ T cells, the transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in vivo. These transcription factors were down-regulated by IMiDs in our in vitro T cell exhaustion analyses. The prevention of excessive T cell exhaustion may maintain effective anti-tumor immunity to cure mature B cell neoplasms.

The cellular and molecular basis of CD69 function in anti-tumor immunity.

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.

IFNγ suppresses the expression of GFI1 and thereby inhibits Th2 cell proliferation.

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 the Gfi1 gene 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.

CD1d expression in glioblastoma is a promising target for NKT cell-based cancer immunotherapy.

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.

Essential Role for CD30-Transglutaminase 2 Axis in Memory Th1 and Th17 Cell Generation.

Memory helper T (Th) cells are crucial for secondary immune responses against infectious microorganisms but also drive the pathogenesis of chronic inflammatory diseases. Therefore, it is of fundamental importance to understand how memory T cells are generated. However, the molecular mechanisms governing memory Th cell generation remain incompletely understood. Here, we identified CD30 as a molecule heterogeneously expressed on effector Th1 and Th17 cells, and CD30hi effector Th1 and Th17 cells preferentially generated memory Th1 and Th17 cells. We found that CD30 mediated signal induced Transglutaminase-2 (TG2) expression, and that the TG2 expression in effector Th cells is essential for memory Th cell generation. In fact, Cd30-deficiency resulted in the impaired generation of memory Th1 and Th17 cells, which can be rescued by overexpression of TG2. Furthermore, transglutaminase-2 (Tgm2)-deficient CD4 T cells failed to become memory Th cells. As a result, T cells from Tgm2-deficient mice displayed impaired antigen-specific antibody production and attenuated Th17-mediated allergic responses. Our data indicate that CD30-induced TG2 expression in effector Th cells is essential for the generation of memory Th1 and Th17 cells, and that CD30 can be a marker for precursors of memory Th1 and Th17 cells.

Overexpression of miR-669m inhibits erythroblast differentiation.

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.

Activated invariant natural killer T cells directly recognize leukemia cells in a CD1d-independent manner.

Invariant natural killer T (iNKT) cells are innate-like CD1d-restricted T cells that express the invariant T cell receptor (TCR) composed of Vα24 and Vß11 in humans. iNKT cells specifically recognize glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d-positive tumor cells, especially when CD1d presents glycolipid antigens. However, iNKT cell recognition of CD1d-negative tumor cells is unknown, and direct cytotoxicity of iNKT cells toward CD1d-negative tumor cells remains controversial. Here, we demonstrate that activated iNKT cells recognize leukemia cells in a CD1d-independent manner, however still in a TCR-mediated way. iNKT cells degranulated and released Th1 cytokines toward CD1d-negative leukemia cells (K562, HL-60, REH) as well as αGalCer-loaded CD1d-positive Jurkat cells. The CD1d-independent cytotoxicity was enhanced by natural killer cell-activating receptors such as NKG2D, 2B4, DNAM-1, LFA-1 and CD2, but iNKT cells did not depend on these receptors for the recognition of CD1d-negative leukemia cells. In contrast, TCR was essential for CD1d-independent recognition and cytotoxicity. iNKT cells degranulated toward patient-derived leukemia cells independently of CD1d expression. iNKT cells targeted myeloid malignancies more than acute lymphoblastic leukemia. These findings reveal a novel anti-tumor mechanism of iNKT cells in targeting CD1d-negative tumor cells and indicate the potential of iNKT cells for clinical application to treat leukemia independently of CD1d.

A new therapeutic target: the CD69-Myl9 system in immune responses.

CD69 is an activation marker on leukocytes. Early studies showed that the CD69+ cells were detected in the lung of patients with asthmatic and eosinophilic pneumonia, suggesting that CD69 might play crucial roles in the pathogenesis of such inflammatory diseases, rather than simply being an activation marker. Intensive studies using mouse models have since clarified that CD69 is a functional molecule regulating the immune responses. We discovered that Myosin light chain 9, 12a, 12b (Myl9/12) are ligands for CD69 and that platelet-derived Myl9 forms a net-like structure (Myl9 nets) that is strongly detected inside blood vessels in inflamed lung. CD69-expressing activated T cells attached to the Myl9 nets can thereby migrate into the inflamed tissues through a system known as the CD69-Myl9 system. In this review, we summarize the discovery of the CD69-Myl9 system and discuss how this system is important in inflammatory immune responses. In addition, we discuss our recent finding that CD69 controls the exhaustion status of tumor-infiltrating T cells and that the blockade of the CD69 function enhances anti-tumor immunity. Finally, we discuss the possibility of CD69 as a new therapeutic target for patients with intractable inflammatory disorders and tumors.

ACC1 determines memory potential of individual CD4+ T cells by regulating de novo fatty acid biosynthesis.

Immunological memory is central to adaptive immunity and protection from disease. Changing metabolic demands as antigen-specific T cells transition from effector to memory cells have been well documented, but the cell-specific pathways and molecules that govern this transition are poorly defined. Here we show that genetic deletion of ACC1, a rate-limiting enzyme in fatty acid biosynthesis, enhances the formation of CD4+ T memory cells. ACC1-deficient effector helper T (Th) cells have similar metabolic signatures to wild-type memory Th cells, and expression of the gene encoding ACC1, Acaca, was inversely correlated with a memory gene signature in individual cells. Inhibition of ACC1 function enhances memory T cell formation during parasite infection in mice. Using single-cell analyses we identify a memory precursor-enriched population (CCR7hiCD137lo) present during early differentiation of effector CD4+ T cells. Our data indicate that fatty acid metabolism directs cell fate determination during the generation of memory CD4+ T cells.

SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity.

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.

Crucial role of CD69 in anti-tumor immunity through regulating the exhaustion of tumor-infiltrating T cells.

The introduction of immune checkpoint inhibitors in cancer treatment highlights the negative regulation of anti-tumor immunity, such as effector T-cell exhaustion in the tumor microenvironment. However, the mechanisms underlying the induction and prevention of T-cell exhaustion remain largely unknown. We found that CD69, a type II glycoprotein known to regulate inflammation through T-cell migration and retention in tissues, plays an important role in inducing the exhaustion of tumor-infiltrating T cells. Cd69-/- mice showed reduced tumor growth and metastasis in a 4T1-luc2 murine breast cancer model, in which increased numbers of tumor-infiltrating lymphocytes, relatively little T-cell exhaustion, and enhanced IFNγ production were observed. Anti-CD69 monoclonal antibody treatment attenuated the T-cell exhaustion and tumor progression in tumor-bearing mice. These findings highlight a novel role of CD69 in controlling the tumor immune escape mediated by T-cell exhaustion and indicate that CD69 is a novel target for cancer immunotherapy.

Anti-tumor immunity via the superoxide-eosinophil axis induced by a lipophilic component of Mycobacterium lipomannan.

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.

Imbalanced expression of polycistronic miRNA in acute myeloid leukemia.

miR-1 and miR-133 are clustered on the same chromosomal loci and are transcribed together as a single transcript that is positively regulated by ecotropic virus integration site-1 (EVI1). Previously, we described how miR-133 has anti-tumorigenic potential through repression of EVI1 expression. It has also been reported that miR-1 is oncogenic in the case of acute myeloid leukemia (AML). Here, we show that expression of miR-1 and miR-133, which have distinct functions, is differentially regulated between AML cell lines. Interestingly, the expression of miR-1 and EVI1, which binds to the promoter of the miR-1/miR-133 cluster, is correlative. The expression levels of TDP-43, an RNA-binding protein that has been reported to increase the expression, but inhibits the activity, of miR-1, were not correlated with expression levels of miR-1 in AML cells. Taken together, our observations raise the possibility that the balance of polycistronic miRNAs is regulated post-transcriptionally in a hierarchical manner possibly involving EVI1, suggesting that the deregulation of this balance may play some role in AML cells with high EVI1 expression.

miRNAs in Normal and Malignant Hematopoiesis.

Lineage specification is primarily regulated at the transcriptional level and lineage-specific transcription factors determine cell fates. MicroRNAs (miRNAs) are 18-24 nucleotide-long non-coding RNAs that post-transcriptionally decrease the translation of target mRNAs and are essential for many cellular functions. miRNAs also regulate lineage specification during hematopoiesis. This review highlights the roles of miRNAs in B-cell development and malignancies, and discusses how miRNA expression profiles correlate with disease prognoses and phenotypes. We also discuss the potential for miRNAs as therapeutic targets and diagnostic tools for B-cell malignancies.

Integrative Network Analysis Combined with Quantitative Phosphoproteomics Reveals Transforming Growth Factor-beta Receptor type-2 (TGFBR2) as a Novel Regulator of Glioblastoma Stem Cell Properties.

Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-ß receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-ß receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-ß receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation.

Thr160 of Axin1 is critical for the formation and function of the ß-catenin destruction complex.

Upon binding of a Wnt ligand to the frizzled (FZD)-low density lipoprotein receptor related protein 5/6 (LRP5/6) receptor complex, the ß-catenin destruction complex, composed of Axin1, adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), is immediately inactivated, which causes ß-catenin stabilization. However, the molecular mechanism of signal transduction from the receptor complex to the ß-catenin destruction complex is controversial. Here we show that Wnt3a treatment promotes the dissociation of the Axin1-APC complex in glioblastoma cells cultured in serum-free medium. Experiments with the GSK3 inhibitor BIO suggest that Axin1-APC dissociation was controlled by phosphorylation. Introduction of a phosphomimetic mutation into Thr160 of Axin1, located in the APC-binding region RGS, abrogated the interaction of Axin1 with APC. Consistent with these observations, the Axin1 phosphomimetic mutant lost the ability to reduce ß-catenin stability and to repress ß-catenin/TCF-dependent transcription. Taken together, our results suggest a novel mechanism of Wnt signaling through the dissociation of the ß-catenin destruction complex by Axin1 Thr160 modification.

SOX9-mediated upregulation of LGR5 is important for glioblastoma tumorigenicity.

LGR5 plays an important role in the self-renewal of stem cells and is used as a marker identifying self-renewing stem cells in small intestine and hair follicles. Moreover, LGR5 has been reported to be overexpressed in several cancers. SOX9 is a transcription factor that plays a key role in development, differentiation and lineage commitment in various tissues. It has also been reported that SOX9 is overexpressed in a variety of cancers and contributes to their malignant phenotype. Here we show that LGR5 is required for the tumorigenicity of glioblastoma cells. We further show that SOX9 is upregulated in glioblastoma cells and directly enhances the expression of LGR5. We also demonstrate that knockdown of SOX9 suppresses the proliferation and tumorigenicity of glioblastoma cells. These results suggest that SOX9-mediated transcriptional regulation of LGR5 is critical for the tumorigenicity of glioblastoma cells. We speculate that the SOX9-LGR5 pathway could be a potentially promising target for the therapy of glioblastoma.

5-Hydroxymethylcytosine plays a critical role in glioblastomagenesis by recruiting the CHTOP-methylosome complex.

The development of cancer is driven not only by genetic mutations but also by epigenetic alterations. Here, we show that TET1-mediated production of 5-hydroxymethylcytosine (5hmC) is required for the tumorigenicity of glioblastoma cells. Furthermore, we demonstrate that chromatin target of PRMT1 (CHTOP) binds to 5hmC. We found that CHTOP is associated with an arginine methyltransferase complex, termed the methylosome, and that this promotes the PRMT1-mediated methylation of arginine 3 of histone H4 (H4R3) in genes involved in glioblastomagenesis, including EGFR, AKT3, CDK6, CCND2, and BRAF. Moreover, we found that CHTOP and PRMT1 are essential for the expression of these genes and that CHTOP is required for the tumorigenicity of glioblastoma cells. These results suggest that 5hmC plays a critical role in glioblastomagenesis by recruiting the CHTOP-methylosome complex to selective sites on the chromosome, where it methylates H4R3 and activates the transcription of cancer-related genes.

PCDH10 is required for the tumorigenicity of glioblastoma cells.

Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell-cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma.