Background: Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Methods and results: Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdc^em26Il2rg^em26Nju (NCG) mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing. Conclusion: We have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody) immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia.

Background CD4⁺ T cells are critical effectors of anti-tumor immunity, but how tumor cells influence CD4⁺ T cell effector function is not fully understood. Tumor cell-released autophagosomes (TRAPs) are being recognized as critical modulators of host anti-tumor immunity during tumor progression. Here, we explored the mechanistic aspects of TRAPs in the modulation of CD4⁺ T cells in the tumor microenvironment. Methods TRAPs isolated from tumor cell lines and pleural effusions or ascites of cancer patients were incubated with CD4⁺ T cells to examine the function and mechanism of TRAPs in CD4⁺ T cell differentiation and function. TRAPs-elicited CD4⁺ T cells were tested for their suppression of effector T cell function, induction of regulatory B cells, and promotion of tumorigenesis and metastasis in a mouse model. Results Heat shock protein 90α (HSP90α) on the surface of TRAPs from malignant effusions of cancer patients and tumor cell lines stimulated CD4⁺ T cell production of IL-6 via a TLR2–MyD88–NF-κB signal cascade. TRAPs-induced autocrine IL-6 further promoted CD4⁺ T cells secretion of IL-10 and IL-21 via STAT3. Notably, TRAPs-elicited CD4⁺ T cells inhibited CD4⁺ and CD8⁺ effector T cell function in an IL-6- and IL-10-dependent manner and induced IL-10-producing regulatory B cells (Bregs) via IL-6, IL-10 and IL-21, thereby promoting tumor growth and metastasis. Consistently, inhibition of tumor autophagosome formation or IL-6 secretion by CD4⁺ T cells markedly retarded tumor growth. Furthermore, B cell or CD4⁺ T cell depletion impeded tumor growth by increasing effector T cell function. Conclusions HSP90α on the surface of TRAPs programs the immunosuppressive functions of CD4⁺ T cells to promote tumor growth and metastasis. TRAPs or their membrane-bound HSP90α represent important therapeutic targets to reverse cancer-associated immunosuppression and improve immunotherapy.

Objectives: MicroRNAs are powerful regulators in hepatocellular carcinoma (HCC) tumorigenesis. MicoRNA‐191 (miR‐191) has been reported to play an important role in HCC, However, the regulatory mechanism is still unclear. In this study, we investigated the role of miR‐191 in HCC and studied its underlying mechanisms of action. Materials and methods: The expression of miR‐191 in HCC tissues was determined by quantitative real‐time PCR (qRT‐PCR). The role of miR‐191 in HCC cells was examined by using both in vitro and in vivo assays. Downstream targets of miR‐191 were determined by qRT‐PCR and Western blot analysis. Dual‐luciferase assays were performed to validate the interaction between miR‐191 and its targets. Results: The expression of miR‐191 was significantly higher in HCC patients and a higher miR‐191 expression predicted poorer prognosis. Analysis of The Cancer Genome Atlas data sets suggested that miR‐191 positively correlated with cell cycle progression. Gain and loss of function assays showed that miR‐191 promoted cell cycle progression and proliferation. Luciferase reporter assay showed that miR‐191 directly targeted the 3'‐untranslated region of KLF6 mRNA. Furthermore, circular RNA has_circ_0000204 could sponge with miR‐191, resulting in inactivation of miR‐191. Conclusions: Our study sheds light on the novel underlying mechanism of miR‐191 in HCC, which may accelerate the development of cancer therapy.

Objectives: Circular RNA, a type of RNA formed by a covalently closed loop, possesses sophisticated abilities of gene regulation in tumorigenesis and metastasis. However, the role of circRNAs on lung adenocarcinoma (LUAD) remains largely unknown.Materials and methods: The role of cMras was examined both in vitro and in vivo. cMras expression in LUAD tissues was determined by quantitative real‐time PCR (qRT‐PCR). Downstream targets of cMras were predicted by bioinformatics tools and confirmed by RNA immunoprecipitation assay and luciferase assay. qRT‐PCR and western blot assay were used to detect the expression of specific targets.Results: Thirty‐six paired LUAD and healthy tissues were collected and cMras resulted significantly downregulated in cancerous tissues. Its expression was negatively associated with tumour stages. cMras overexpression suppressed LUAD growth and metastasis, while endogenous cMras silencing resulted in the opposite effects. Bioinformatics analysis and experimental evidence confirmed that cMras was a sponge of miRNA‐567 and released its direct target, PTPRG. cMras overexpression decreased miR‐567 expression and subsequently increased PTPRG expression, while increased miRNA‐567 expression blocked the effects induced by cMras. Moreover, PTPRG was downregulated in LUAD and patients with low PTPRG expression exhibited significantly poor prognosis. These results suggested that cMras/miR‐567/PTPRG regulatory pathway might be associated to LUAD tumorigenesis and development.Conclusions: A novel circular RNA cMras and its functions were identified, discovering a cMras/miR‐567/PTPRG regulatory pathway in LUAD tumorigenesis and development.

Dendritic cells (DCs) regulate innate and acquired immunity through their roles as antigen-presenting cells. Specific subsets of mature DCs, including monocyte-derived and lymphoid-derived DCs, can be distinguished based on distinct immunophenotypes and functional properties. The leukocyte integrin, CD11c, is considered a specific marker for DCs and it is expressed by all DC subsets. We created a strain of mice in which DCs and their progenitors could be lineage traced based on activity of the CD11c proximal promoter. Surprisingly, we observed levels of CD11c promoter activity that were similar in DCs and in other mature leukocytes, including monocytes, granulocytes, and lymphocytes. We sought to identify DNA elements and transcription factors that regulate DC-associated expression of CD11c. The ets transcription factor, PU.1, is a key regulator of DC development, and expression of PU.1 varies in different DC subsets. GM-CSF increased monocyte-derived DCs in mice and from mouse bone marrow cultured in vitro, but it did not increase CD8⁺ lymphoid-derived DCs or B220⁺ plasmacytoid DCs. FLT3L increased both monocyte-derived DCs and lymphoid-derived DCs from mouse bone marrow cultured in vitro. GM-CSF increased the 5.3 Kb CD11c proximal promoter activity in monocyte-derived DCs and CD8⁺ lymphoid-derived DCs, but not in B220⁺ plasmacytoid DCs. In contrast, FLT3L increased the CD11c proximal promoter activity in both monocyte-derived DCs and B220⁺ plasmacytoid DCs. We used shRNA gene knockdown and chromatin immunoprecipitation to demonstrate that PU.1 is required for the effects of GM-CSF or FLT3L on monocyte-derived DCs. We conclude that both GM-CSF and FLT3L act through PU.1 to activate the 5.3 Kb CD11c proximal promoter in DCs and to induce differentiation of monocyte-derived DCs. We also confirm that the CD11c proximal promoter is not sufficient to direct lineage specificity of CD11c expression, and that additional DNA elements are required for lineage-specific CD11c expression.