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Interleukin-12 (IL-12) has potent antitumor activity, but its clinical application is limited by severe systemic toxicity, which might be alleviated by the use of membrane-anchored IL-12. In the present study, a new membrane-bound IL-12 containing murine single-chain IL-12 and B7-1 transmembrane and cytoplasmic domains (scIL-12-B7TM) was constructed and its efficacy in cancer treatment examined and its protective antitumor mechanism investigated. Surface expression of scIL-12-B7TM on colon adenocarcinoma cells significantly inhibited the growth of subcutaneous tumors, suppressed lung metastasis, and resulted in local and systemic suppression of unmodified tumors. Intratumoral injection of an adenoviral vector encoding scIL-12-B7TM not only resulted in complete regression of a majority of local tumors, but also significantly suppressed the growth of distant, untreated tumors. Moreover, mice that had been treated with scIL-12-B7TM developed memory responses against subsequent tumor challenge. Immunohistochemical staining and in vivo depletion of lymphocyte subpopulations demonstrated that both CD8(+) T cells and CD4(+) T cells contributed to the antitumor activity of scIL-12-B7TM. Importantly, the potent antitumor activities of scIL-12-B7TM were achieved with only negligible amounts of IL-12 in the circulation. Our data demonstrate that cancer immunotherapy using membrane-bound IL-12 has the advantage of minimizing systemic IL-12 levels without compromising its antitumor efficacy.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, but effective therapies are still needed. The liver has been identified as an important immune organ and is heavily populated with various lymphocyte subsets known to play important roles in cancer immunosurveillance. We hypothesized that activation of hepatic lymphocytes by interleukin (IL)-15, a cytokine known for its ability to trigger proliferation and activation of natural killer (NK) cells, natural killer T cells, and memory CD8(+) T cells, might offer an alternative therapy for HCC. We employed hepatotropic adeno-associated virus serotype 8 (AAV8) to deliver an IL-15 superagonist (IL-15-IL-15RalphaS), consisting of IL-15 covalently linked to the N-terminal sushi domain of the IL-15 receptor alpha chain, to achieve local sustained cytokine expression in the liver environment. We observed that a single injection of AAV8 expressing IL-15-IL-15RalphaS, but not IL-15 alone, greatly expanded the number of hepatic mononuclear cells, mainly NK cells, for at least 21 days. AAV8/IL-15-IL-15RalphaS treatment generated potent antitumor activity in a liver metastatic murine HCC model (BNL cells), and significantly prolonged the survival time of treated animals. The antitumor effect depended mainly on NK cells, not on CD8(+) and CD4(+) T cells, because AAV8/IL-15-IL-15RalphaS treatment greatly enhanced the cytolytic activity of hepatic NK cells and depletion of NK cells abrogated the therapeutic effect. Importantly, no apparent liver toxicity was observed during AAV8/IL-15-IL-15RalphaS treatment. Together, our data demonstrate that AAV8-delivered IL-15-IL-15RalphaS provides an effective and safe therapy against metastatic HCC.

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 ± 333 mm3, then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8+ T cells, but not CD4+ T cells or NK cells, were crucial for the antitumor activity of IL-23.

Galectin-3 has been reported to regulate the functions of a number of immune cell types. We previously reported that galectin-3 is translocated to immunological synapses in T cells upon T-cell receptor engagement, where it associates with ALG-2-interacting protein X (Alix). Alix is known to coordinate with the endosomal sorting complex required for transport (ESCRT) to promote human immunodeficiency virus (HIV)-1 virion release. We hypothesized that galectin-3 plays a role in HIV-1 viral budding. Cotransfection of cells of the Jurkat T line with galectin-3 and HIV-1 plasmids resulted in increased HIV-1 budding, and suppression of galectin-3 expression by RNAi in Hut78 and primary CD4+ T cells led to reduced HIV-1 budding. We used immunofluorescence microscopy to observe the partial colocalization of galectin-3, Alix and Gag in HIV-1-infected cells. Results from co-immunoprecipitation experiments indicate that galectin-3 expression promotes Alix-Gag p6 association, whereas the results of Alix knockdown suggest that galectin-3 promotes HIV-1 budding through Alix. HIV-1 particles released from galectin-3-expressing cells acquire the galectin-3 protein in an Alix-dependent manner, with proteins primarily residing inside the virions. We also found that the galectin-3 N-terminal domain interacts with the proline-rich region of Alix. Collectively, these results suggest that endogenous galectin-3 facilitates HIV-1 budding by promoting the Alix-Gag p6 association.

Galectin-7, a member of the β-galactoside-binding protein family, is primarily expressed in stratified epithelial cells, including keratinocytes. There is information in the literature suggesting a role for this protein in regulation of keratinocyte survival and growth, but the underlying mechanism remains relatively unknown. Moreover, its expression pattern in the epidermis suggests that it is also involved in the regulation of keratinocyte differentiation. Here, we demonstrate that galectin-7 knockdown results in reduced differentiation and increased proliferation of keratinocytes. Using microarray and deep-sequencing analyses, we found that galectin-7 positively and negatively regulates microRNA (miR)-203 and miR-146a expression, respectively. We show that galectin-7 regulates keratinocyte differentiation and proliferation through miR-203 but not miR-146a. A knockdown of either galectin-7 or miR-203 in keratinocytes increases expression of p63, an essential transcription factor involved in skin development. Rescue of miR-203 expression in a galectin-7 knockdown model reduces p63 expression to baseline. Increased galectin-7 expression up-regulates c-Jun N-terminal kinase (JNK) protein levels, which is required for miR-203 expression. Finally, we establish that galectin-7 can be associated with JNK1 and protect it from ubiquitination and degradation. Thus, our data suggest an intracellular function of galectin-7: regulation of keratinocyte proliferation and differentiation through the JNK1-miR-203-p63 pathway.