Yutaka Tagaya

Expression and growth-promoting effect of adult t-cell leukemia-derived factor a human thioredoxin homologue in hepatocellular carcinoma

Adult T‐cell leukemia‐derived factor (ADF), originally defined as an interleukin‐2 receptor inducer, is a human thioredoxin homologue. ADF is detected in many malignant tissues and has a growth‐promoting effect on transformed cells. In this study, ADF expression was examined immunohistochemically in human liver cell lines and liver tissues, and its growth‐promoting effect was tested on human hepatoma cells. On three liver cell lines—PLC/PRF/5, HepG2, and Chang liver cells—ADF stained positively and also was detected by immunoblotting. ADF had strong staining in the fetal liver (n = 8), although it was faint in the normal adult liver (n = 6). In hepatocellular carcinoma (n = 25), ADF expression generally was enhanced and was very strong in 52% (13 of 25) of the cases, although it was moderate in cases of chronic hepatitis or cirrhosis. ADF augmented the growth of PLC/PRF/5 cells and showed an additive effect with epidermal growth factor. These results indicate possible involvement of ADF in cell activation and growth of hepatocytes, as is the case with lymphocytes.

The IL-15/IL-15Rα on cell surfaces enables sustained IL-15 activity and contributes to the long survival of CD8 memory T cells

We previously described unique features of the IL-15 receptor (IL-15R)α. IL-15Rα by itself forms stable complexes with IL-15 on cell surfaces and presents IL-15 in trans to neighboring natural killer/T cells. Moreover, the membrane IL-15/IL-15Rα complexes (membIL-15) undergo endosomal internalization but survive lysosomal degradation, allowing the complexes to recycle back to the cell surface. Here, we show that membIL-15+ cells act as a persistent source of IL-15 for the surrounding microenvironment (intercellular reservoir effect). Additionally, membIL-15+ cells give rise to augmented retention of IL-15 in the circulation as well as in tissues. Curiously, IL-15 retention was particularly associated with lungs, rather than with lymph nodes, in normal unstimulated mice, which correlated with the preferential homing of antigen-specific CD8 T cells to lungs during their contraction phase in an IL-15Rα-dependent manner. Furthermore, membIL-15, unlike soluble IL-15, caused sustained IL-15 signal transduction in the target cells. Collectively, these characteristics define IL-15 as a unique cytokine with prolonged in vivo survival and sustained biological action on the target cells, which may account for the proposed persistent action of IL-15 that helps the long-term survival of functional CD8 memory T cells in vivo.

HIV infection-associated immune activation occurs by two distinct pathways that differentially affect CD4 and CD8 T cells

HIV infection is characterized by a brisk immune activation that plays an important role in the CD4 depletion and immune dysfunction of patients with AIDS. The mechanism underlying this activation is poorly understood. In the current study, we tested the hypothesis that this activation is the net product of two distinct pathways: the inflammatory response to HIV infection and the homeostatic response to CD4 T cell depletion. Using ex vivo BrdU incorporation of PBMCs from 284 patients with different stages of HIV infection, we found that CD4 proliferation was better predicted by the combination of CD4 depletion and HIV viral load (R2 = 0.375, P < 0.001) than by either parameter alone (CD4 T cell counts, R2 = 0.202, P < 0.001; HIV viremia, R2 = 0.302, P < 0.001). Interestingly, CD8 T cell proliferation could be predicted by HIV RNA levels alone (R2 = 0.334, P < 0.001) and this predictive value increased only slightly (R2 = 0.346, P < 0.001) when CD4 T cell depletion was taken into account. Consistent with the hypothesis that CD4 T cell proliferation is driven by IL-7 as a homeostatic response to CD4 T cell depletion, levels of phosphorylated STAT-5 were found to be elevated in naive subsets of CD4 and CD8 T cells from patients with HIV infection and in the central memory subset of CD4 T cells. Taken together these data demonstrate that at least two different pathways lead to immune activation of T cells in patients with HIV infection and these pathways differentially influence CD4 and CD8 T cell subsets.

Acquisition of CD80 (B7-1) by T cells

Activation of T cells usually requires two signals. Signal 1 is mediated via a peptide-MHC on the APC; signal 2 is mediated via a costimulatory molecule on the APC surface. We demonstrate here that naive CD4+ T cells actually acquire the costimulatory molecule CD80 (B7-1) from syngeneic APCs after activation. This phenomenon was demonstrated showing acquisition of CD80 by T cells from CD80/CD86 (B7-2) knockout mice, and by treating T cells with cyclohexamide to further rule out endogenous expression of CD80 by T cells. Moreover, no CD80 mRNA could be detected in T cells that had acquired CD80. The amount of acquisition of CD80 by T cells was shown to be directly related to both the strength of signal 1 and the amount of CD80 on the APC. Specificity of this acquisition was also shown by the lack of acquisition by T cells from CD28 knockout mice (implicating CD28 in this process), the lack of acquisition of CD40 (another molecule on the APC surface) by T cells, and confocal microscopy studies. We demonstrate for the first time that 1) naive T cells, following acquisition of CD80 from APCs, were themselves shown to be capable of acting as APCs; and 2) memory T cells that have acquired CD80 from APCs undergo apoptosis in the presence of increased levels of signal 1. Thus we demonstrate both immunostimulatory and immunoregulatory functions as a result of CD80 acquisition by different T cell populations.

Tumor-induced impairment of TCR signaling results in compromised functionality of tumor-infiltrating regulatory T cells.

This study demonstrates, for the first time, that murine regulatory T (Treg) cells in the tumor microenvironment display both enhanced proliferation and reduced functionality. This enhanced proliferation, combined with decreased apoptosis, leads to an intratumoral accumulation of Treg cells with a unique phenotype: CD4+CD25+FoxP3+GITRhighCD27lowCD62L−. The loss of functionality is associated with down-regulation of the TCR signaling complex, including IL-2-inducible T cell kinase. It is also demonstrated that tumor-infiltrating Treg cells have impaired TCR-mediated signaling and calcium influx. Based on these findings, this study supports the hypothesis that 1) tumor-infiltrating Treg cells lose functionality due to their diminished ability to become effectively activated and 2) intratumoral accumulation of Treg cells may compensate for the impaired functionality, thus maintaining immune tolerance to the tumor.

Development of an IL-15–autocrine CD8 T-cell leukemia in IL-15–transgenic mice requires the cis expression of IL-15Rα

IL-15 has growth-promoting effects on select lymphoid subsets, including natural killer (NK) cells, NK T cells, intraepithelial lymphocytes (IELs), CD8 T cells, and γδ-T cells. Constitutive expression of murine IL-15 in IL-15-transgenic mice was reported to cause T-NK leukemia. We investigated whether IL-15 expression is sufficient for leukemic transformation using a human IL-15-transgenic (IL-15Tg) mouse model. We noted that 100% of the mice observed over a 2-year period (n > 150) developed fatal expansions of CD8 T cells with NK markers, and determined that these cells expressed IL-15 receptor alpha (IL-15Rα). The expression of IL-15Rα on CD8 T cells appears to be required for uncontrolled aggressive lymphoproliferation, because none of the IL-15Rα(-/-)-IL-15Tg mice that we followed for more than 2 years developed the fatal disease despite controlled expansion of CD8 T cells. In addition, in contrast to IL-15Tg mice, in which leukemia-like CD8 T cells expressed IL-15Rα persistently, acutely activated normal CD8 T cells only transiently expressed IL-15Rα. Inhibition of DNA methylation enabled sustained IL-15Rα expression induced by activation. We present a scenario for IL-15Tg mice in which CD8 T cells that acquire constitutive persistent IL-15Rα expression are at a selective advantage and become founder cells, outgrow other lymphocytes, and lead to the establishment of a leukemia-like condition.

Activation of interleukin-2 receptor gene by Forskolin and cyclic AMP analogues

Forskolin (FK), a reversible activator of adenylate cyclase, markedly enhanced the expression of interleukin-2 receptor (IL-2-R) on a human natural killer (NK)-like cell line, YT. The FK-induced increase in IL-2-R on YT cells closely correlated with an increase in intracellular cyclic AMP (cAMP) level, and was mimicked by dibutyryl cyclic AMP (dbcAMP). FK induced both high and low affinity IL-2-R on the cells. Using a cDNA for the IL-2-R as a probe, the FK-induced IL-2-R expression was shown to be associated with an increase in IL-2-R mRNA. FK also enhanced the IL-2-R expression on a human T lymphotrophic virus I (HTLV-I) positive T-cell line (YTA-1H) and augmented the phorbol ester-induced expression of IL-2-R on HTLV-I negative T-cell lines (HSB-2 and HPB-ALL). These results suggest the possibility that the stimulation of adenylate cyclase may serve as a pathway leading to activation of the IL-2-R gene in certain types of lymphocytes.

Interleukin 1α mRNA in virus-transformed T and B cells

Summary IL-lα cDNA clone was isolated from a T cell line infected by the human T lymphocropic retrovirus type-I (HTLV-I/ATLV). We found significant amounts of mRNA hybridizing to IL-lα cDNA not only in HTLV-I-transformed T cells but also in Epstein-Barr Virus-transformed B cells. A part of IL-2 receptor inducing activity in Adult T cell leukemia (ATL) cell line seems to be due to IL-lα.

IL-15 Expands Unconventional CD8ααNK1.1+ T Cells but Not Vα14Jα18+ NKT Cells

Despite recent gains in knowledge regarding CD1d-restricted NKT cells, very little is understood of non-CD1d-restricted NKT cells such as CD8+NK1.1+ T cells, in part because of the very small proportion of these cells in the periphery. In this study we took advantage of the high number of CD8+NK1.1+ T cells in IL-15-transgenic mice to characterize this T cell population. In the IL-15-transgenic mice, the absolute number of CD1d-tetramer+ NKT cells did not increase, although IL-15 has been shown to play a critical role in the development and expansion of these cells. The CD8+NK1.1+ T cells in the IL-15-transgenic mice did not react with CD1d-tetramer. Approximately 50% of CD8+NK1.1+ T cells were CD8αα. In contrast to CD4+NK1.1+ T cells, which were mostly CD1d-restricted NKT cells and of which ∼70% were CD69+CD44+, ∼70% of CD8+NK1.1+ T cells were CD69−CD44+. We could also expand similar CD8ααNK1.1+ T cells but not CD4+ NKT cells from CD8α+β− bone marrow cells cultured ex vivo with IL-15. These results indicate that the increased CD8ααNK1.1+ T cells are not activated conventional CD8+ T cells and do not arise from conventional CD8αβ precursors. CD8ααNK1.1+ T cells produced very large amounts of IFN-γ and degranulated upon TCR activation. These results suggest that high levels of IL-15 induce expansion or differentiation of a novel NK1.1+ T cell subset, CD8ααNK1.1+ T cells, and that IL-15-transgenic mice may be a useful resource for studying the functional relevance of CD8+NK1.1+ T cells.

Transcription of IL-2 receptor gene is stimulated by ATL-derived factor produced by HTLV-I(+) T cell lines

In HTLV-I transformed T-cell lines established from the patients with adult T-cell leukemia (ATL), there is a constitutive activation of the normal IL-2 receptor (IL-2-R) gene. These cell lines continuously produce an ATL-derived factor (ADF), an IL-2-R inducing factor without IL-2 activity. ADF enhances the expression of the IL-2-R through the augmentation of the IL-2-R mRNA in the HTLV-I(+) T-cell line (ED) as well as the NK cell line cells (YT). In YT cells, the transcriptional initiation of the promoter of the IL-2-R gene was enhanced by ADF but not by IL-2. Production of ADF by HTLV-I(+) T-cell lines may be involved in the abnormal expression of IL-2-Rs on these cells.