Takeshi Otani

IFN-γ-Dependent and -Independent Mechanisms in Adverse Effects Caused by Concomitant Administration of IL-18 and IL-12

IL-18 is a new type of inflammatory cytokine similar to but distinct from IL-12 and IL-1β. One intriguing property of IL-18 is synergism with IL-12 in many respects. In this study we examined the in vivo synergistic effects of IL-18/IL-12 in mice and found lethal toxicity accompanying an elevated IFN-γ level in the serum. Since treatment with IL-18 alone did not have any apparent toxicity, and treatment with IL-12 alone showed only limited toxicity in our system, the synergy between the two cytokines was all the more remarkable. The major symptoms of the toxicity were weight loss, diarrhea, hemorrhagic colitis, splenomegaly, fatty liver, and atrophic thymus, most of which are similarly found in endotoxin-induced septic shock. However, in contrast to septic shock, TNF-α was not induced. The involvement of IFN-γ in the toxicity was further studied in detail. Treatment of athymic nude mice with anti-asialo-GM1 did not reduce the toxicity, whereas anti-IFN-γ treatment of wild-type mice alleviated it. When IFN-γ-deficient mice were treated with IL-18/IL-12, the majority of them showed mortality and toxicity with severe pulmonary edema. These results indicate that IL-18/IL-12 treatment induces severe adverse effects through not only IFN-γ-dependent mechanisms but also IFN-γ-independent processes.

The Production of IL-10 by Human Regulatory T Cells Is Enhanced by IL-2 through a STAT5-Responsive Intronic Enhancer in the IL-10 Locus

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4+CD25+ naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.

miR-155, a Modulator of FOXO3a Protein Expression, Is Underexpressed and Cannot Be Upregulated by Stimulation of HOZOT, a Line of Multifunctional Treg.

MicroRNAs (miRNAs) play important roles in regulating post-transcriptional gene repression in a variety of immunological processes. In particular, much attention has been focused on their roles in regulatory T (Treg) cells which are crucial for maintaining peripheral tolerance and controlling T cell responses. Recently, we established a novel type of human Treg cell line, termed HOZOT, multifunctional cells exhibiting a CD4+CD8+ phenotype. In this study, we performed miRNA profiling to identify signature miRNAs of HOZOT, and therein identified miR-155. Although miR-155 has also been characterized as a signature miRNA for FOXP3+ natural Treg (nTreg) cells, it was expressed quite differently in HOZOT cells. Under both stimulatory and non-stimulatory conditions, miR-155 expression remained at low levels in HOZOT, while its expression in nTreg and conventional T cells remarkably increased after stimulation. We next searched candidate target genes of miR-155 through bioinformatics, and identified FOXO3a, a negative regulator of Akt signaling, as a miR-155 target gene. Further studies by gain- and loss-of-function experiments supported a role for miR-155 in the regulation of FOXO3a protein expression in conventional T and HOZOT cells.

Cell-in-cell Structures Formed between Human Cancer Cell Lines and the Cytotoxic Regulatory T-cell Line HOZOT

We previously established a novel cell line, termed HOZOT, derived from umbilical cord blood mononuclear cells that is characterized as a human cytotoxic regulatory T (Treg) cell line with a FOXP3(+)CD4(+)CD8(+)CD25(+) phenotype. Here, we describe a new property of HOZOT cells: they actively penetrate into a variety of human cancer cell lines, but not into normal cell lines, and form apparent cell-in-cell structures. In the process of cell penetration, we observed that HOZOT cells adhered to target cells seemed to first insert their nuclei into the cytoplasm of target cells, distinct from the process of phagocytosis. In addition, blocking experiments showed that major histocompatibility complex class I is one of the target cell recognition molecules for HOZOT cells. Furthermore, we propose that cell-in-cell structures between HOZOT cells and target cancer cells could be one of the cytotoxic mechanisms of HOZOT cells.

Novel mechanisms of suppressor activity exhibited by cytotoxic regulatory T cell lines, HOZOT

OBJECTIVE Regulatory T (Treg) cells, which play a central role in maintaining immune tolerance, can be grouped into different subtypes, such as naturally occurring Treg, type-1 T regulatory, and Th3. The suppressor activities of Treg cells are mediated through several molecular mechanisms, including immunosuppressive cytokines, cell surface molecules, and cytolytic molecules. In a previous report, we described a novel regulatory human T-cell line (termed HOZOT). The line was established by cocultivating human umbilical cord blood cells with mouse stromal cells in the absence of exogenous cytokines. In this study, we investigated the mechanism of HOZOTs suppressor activity. MATERIALS AND METHODS Suppressor activity of HOZOT was evaluated in vitro by assessing their inhibition of allogeneic mixed lymphocyte reaction, in which CD4+CD25(-) responder T cells were stimulated by dendritic cells (DCs). Responder T cells as well as DCs were prepared from umbilical cord blood using magnetic-activated cell sorting separation system. DNA microarray analysis was performed to search for specific molecules involved in HOZOTs suppressor mechanisms. RESULTS We confirmed that suppressing effects were observed in all three subpopulations of CD4/CD8 phenotype. We ruled out possible involvement of HOZOTs cytotoxic activity as well as participation of surface molecules, including cytotoxic T-lymphocyte-associated protein-4, programmed death-1, and glucocorticoid-inducible tumor necrosis factor receptor in suppressor. The supernatant obtained from HOZOT and DC coculture revealed mixed lymphocyte reaction inhibitory activity, indicating the presence of a soluble factor, which mediates suppressor function. Blocking experiments demonstrated that interleukin-10 and transforming growth factor-beta were not responsible factors. CONCLUSIONS HOZOT exerted suppressor activity in the absence of cell contact mechanisms, which are distinct from those of naturally occurring Treg, type-1 T regulatory, and Th3.

Interleukin-8 and RANTES are signature cytokines made by HOZOT, a new type of regulatory T cells

Distinct cytokine production profiles define the effector functions of both helper and regulatory T cells. Recently, we established novel cytotoxic regulatory T (Treg) cell lines, HOZOT, which have been characterized as IL-10-producing T cells. In this study, we further characterized HOZOT by performing comprehensive analyses of cytokines produced by HOZOTs in order to identify a signature cytokine profile. Using DNA microarrays, we compared the gene expression profiles of HOZOT-4, a representative HOZOT cell line, under three different conditions. Seven genes, including IL-8, IL-10, IL-13, MIP-1alpha, and MIP-1beta, were identified as inducible cytokines when stimulated with stromal cells or anti-CD3/CD28 antibodies. Twelve genes, including IL-2, IL-3, IL-4, IL-22, CCL1, and lymphotactin, were categorized as antibody stimulation-responsive but stromal cell-non-responsive. Three genes, IL-32, RANTES, and CCL23, were constitutively expressed irrespective of stimulation condition. Among these cytokines, we focused on two chemokines, IL-8 and RANTES for further studies, and found that only HOZOT produced both of them at considerable levels whereas other T cell subsets, including Tregs and helper T cells, did not. Kinetic and inhibition experiments revealed contrasting properties for the two chemokines. IL-8 was induced only after stimulation, whereas RANTES mRNA and protein accumulated to high levels even before stimulation. Interestingly, IL-8 mRNA was induced by cycloheximide treatment and RANTES showed reduced mRNA but increased protein expression by antibody stimulation. As a whole, the unique cytokine signature profile consisting of Th1, Th2, and cytolytic T cell cytokines as well as Treg cytokines reflect the multifunctional nature of HOZOT. In particular, the dual production of IL-8 and RANTES by distinct mechanisms is a hallmark of HOZOT.

HOZOTs, novel human regulatory T-cell lines, exhibit helper or suppressor activities depending on dendritic cell or anti-CD3 stimulation

OBJECTIVE HOZOT cell lines (HOZOTs) are a new type of regulatory T cells established from human umbilical cord blood without using cytokines. In addition to their unique FOXP3(+)CD4(+)CD8(+)CD25(+) phenotype, HOZOTs are bifunctional and can exert either suppressor or cytotoxic activities. To further characterize HOZOTs, we cocultured HOZOTs with responder T cells under different stimulation conditions and found another function of HOZOTs. MATERIALS AND METHODS Naïve CD4(+)T cells as responder cells were stimulated with dendritic cells or plate bound anti-CD3 antibody. As effector cells, HOZOTs were added to this culture and proliferation of the responder cells were monitored by (3)H-thymidine incorporation or carboxyfluorescein succinimidyl ester dilution method. To investigate the molecular mechanisms, antibodies specific for interleukin (IL)-2/IL-2R or cell surface molecules were used for blocking experiments. RESULTS The proliferation of naïve CD4(+)T cells was suppressed by one HOZOT line, HOZOT-4, when the responder cells were stimulated with dendritic cells. However, responder cell proliferation was augmented by HOZOT-4 when these cells were stimulated with anti-CD3 antibody. This opposing function to responder cells was unique to HOZOTs because naturally occurring regulatory T cells suppressed proliferation of both dendritic cell- and anti-CD3-antibody-stimulated cells. IL-2 was not involved in the mechanism of the helper activity of HOZOT-4 as blocking antibodies for IL-2 and IL-2R did not abrogate the helper activity. Moreover, this helper activity could not be reduced by blocking costimulatory pathways such as CD28/B7, CD4/human leukocyte antigen-DR, and intercellular adhesion molecule-1/lymphocyte function-associated antigen-1. CONCLUSION We demonstrated a new function of HOZOTs as helper T cells in addition to suppressor and cytotoxic activities, characterizing HOZOTs as multifunctional T cells.

Potent anti-tumor killing activity of the multifunctional Treg cell line HOZOT against human tumors with diverse origins.

The T cell line HOZOT has a unique FOXP3+CD4+ CD8+CD25+ phenotype, exhibits suppressive activity in allogeneic mixed lymphocyte reactions (MLR), and produces IL-10, defining HOZOT as regulatory T cells (Tregs). Interestingly, in addition to possessing a suppressive Treg ability, HOZOT was also found to show cytotoxicity against certain representative human cancer cell types. In order to disclose the range of anti-tumor activity by HOZOT, we screened it by using a panel of twenty human tumor cell lines with different origins. Consequently, HOZOT showed potent cytocidal effects against a wide spectrum of neoplastic cells including carcinomas, sarcomas, mesotheliomas and glioblastomas except for hematopoietic malignancies. Its anti-tumor activity was strong enough with an E:T ratio of 4:1, which is considered to be more effective than that by conventional CTLs. Furthermore, an in vivo representative mouse tumor model by implanting human colon adenocarcinoma cells revealed that adoptive transfer of HOZOT almost completely eradicated disseminated lesions on peritoneum, markedly reduced metastases in lung and liver, and dramatically decreased bloody ascites caused by peritoneal carcinomatosis. Treatment of the tumor model mice by HOZOT with an E:T ratio of 2:1 even indicated the prolongation of their survival, although not reaching obvious statistical significance. In vitro blocking experiments using antibodies and inhibitors suggested that the cytotoxic mechanism of HOZOT against tumors is different from conventional cytotoxic cells such as CTL, NK or NKT cells. Altogether, our studies demonstrated the potent killing activity of HOZOT against a broad range of human malignancies, which indicates that HOZOT is a powerful tool in immunotherapy for advanced stage tumors.

Marked induction of CD4+CD8+ T cells with multifunctional properties by coculturing CD2+ cells with xenogeneic stromal cells.

A number of T cell subsets have been identified, and the in vitro characterization of these subsets largely depends on an appropriate induction system for each one. In previous studies, we characterized a unique T cell line, HOZOT, which possessed a CD4+CD8+ double positive (DP) phenotype and multifunctional properties including cytotoxic, helper, and regulatory functions. Therefore, this T cell subset has been termed Tchreg cells. In this study, we established a new method of Tchreg cell induction, which consists of three steps and provides more efficient and reproducible results. By using a purified T cell population, the efficiency of Tchreg generation was profoundly increased, and the use of purified CD2+ cells rather than CD3+ cells was shown to be superior. One surprising finding was that at the initial step, stromal cell stimulation induced DP T cells more efficiently than dendritic cells or anti-T cell receptor stimulation, indicating a distinct antigen presenting cell (APC) ability of stromal cells as distinguished from conventional APCs. Even in subsequent steps, the presence of stromal cells was required to maintain the DP phenotype. In the second step, addition of stromal cell-conditioned (but not unconditioned) autologous CD14+ cells instead of interleukin-2 was beneficial for subsequent expansion of Tchreg cells. The improved three-step culture method described here represents a step forward in efforts to achieve clinical application and a good tool for elucidating how Tchreg cells, especially CD4+CD8+ T cells, are generated.

Stromal cells' B7-1 is a key stimulatory molecule for interleukin-10 production by HOZOT, a multifunctional regulatory T-cell line.

We have previously shown that xenogeneic stromal cell stimulation of naïve T cells resulted in the generation of a new type of regulatory T (Treg) cell termed HOZOT, which has multifunctional properties and a CD4/CD8 double‐positive phenotype. Even after the establishment of HOZOT, stromal cells can function as an antigen‐presenting cell (APC) by inducing these cells to produce interleukin (IL)‐10. When compared with other stimuli, stromal cells showed an IL‐10‐producing ability comparable to anti‐CD3 antibody (Ab) stimulation, and much greater than dendritic cell (DC) stimulation. Distinct from professional APCs, stromal cells express only major histocompatibility complex (MHC) class I and B7‐1 costimulatory molecules, and not MHC class II or other costimulatory molecules, such as ICOSL (CD275), PD‐L1 (CD274), PD‐L2 (CD273), CD40, OX40L (CD252) and 4‐1BBL (CD137L) in the absence of stimulation. Blocking experiments revealed that, in addition to anti‐H‐2Kd Ab and anti‐human CD8 Ab, anti‐mouse B7‐1 Ab could effectively block IL‐10 production, indicating a key role of the B7‐1/CD28 pathway. Using stromal cells expressing different levels of B7‐1, IL‐10 production correlated with the levels of B7‐1 expression. Distinct from ICOSL or PD‐L1 expressed on DCs (which are regarded as IL‐10‐inducing costimulatory molecules), this study showed that B7‐1 on stromal cells is a key molecule regulating IL‐10 production by multifunctional Treg cells, HOZOT.