Tumour‐experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)‐γ secretion on engagement of the natural‐killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell‐mediated anti‐tumour responses. Here, we investigated the early events occurring during T cell–tumour cell interactions, and their impact on NK cell functions. We observed that on co‐culture with some melanomas, activated CD4⁺ T cells promoted degranulation, and NKG2D‐ and NKp46‐dependent IFN‐γ secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour‐cell surface (trogocytosis). This effect was observed in CD4⁺, CD8⁺ and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain‐related protein A on co‐culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity.     

CD83+CCR7+ NK cells induced by interleukin 18 by dendritic cells promote experimental autoimmune uveitis

Natural killer (NK) cells have been reported to play a pathological role in autoimmune uveitis. However, the mechanisms regarding NK cells in uveitis and factors that affect NK‐cell activation in this condition remain unclear. Here, we report that the number of CD3^‐NK1.1⁺CD83⁺CCR7⁺ cells is increased in the inflamed eyes within a mouse model of experimental autoimmune uveitis (EAU), and these cells express elevated levels of NKG2D, CD69 and IFN‐γ. Adoptively transferring CD83⁺CCR7⁺NK cells aggravates EAU symptoms and increases the number of CD4⁺IFN‐γ⁺T cells and dendritic cells (DCs) within the eye. These CD83⁺CCR7⁺NK cells then promote the maturation of DCs and IFN‐γ expression within T cells as demonstrated in vitro. Furthermore, IL‐18, as primarily secreted by DCs in the eyes, is detected to induce CD83⁺CCR7⁺NK cells. In EAU mice, anti‐IL‐18R antibody treatment also decreases retinal tissue damage, as well as the number of infiltrating CD83⁺CCR7⁺NK cells, T cells and DCs in the inflamed eyes and spleens of EAU mice. These results suggest that CD83⁺CCR7⁺NK cells, as induced by IL‐18 that primarily secreted by DCs, play a critical pathological role in EAU. Anti‐IL‐18R antibody might serve as a potential therapeutic agent for uveitis through its capacity to inhibit CD83⁺CCR7⁺NK cells infiltration.     

Prostate Tumor-Derived Exosomes Down-Regulate NKG2D Expression on Natural Killer Cells and CD8+ T Cells: Mechanism of Immune Evasion

Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8⁺ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8⁺ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8⁺ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape.     

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1

Background

The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8⁺ cytotoxic T cells and is classically considered absent in CD4⁺ T cells. However, recent studies have identified a distinctive population of CD4⁺ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4⁺ T cell-immunocompromised individuals suggests that CD4⁺ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4⁺ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4⁺NKG2D⁺ T cell expansion were also measured.

Results

Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4⁺ T cells and CD8⁺ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4⁺ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4⁺NKG2D⁺ T cells was seen in patients with CIN 1, despite the overall levels of CD4⁺ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4⁺NKG2D⁺ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group.

Conclusions

Taken together, our results suggest that the significant increase within the CD4⁺NKG2D⁺ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion.     

Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer cells

NKG2D is a surface receptor expressed on NK cells but also on CD8⁺ T cells, γδ T cells, and auto-reactive CD4⁺/CD28⁻ T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (K_D) of 2.7 ± 1.4 × 10⁻¹¹ M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFα, IFNγ and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after seven days in human serum at 37°C, and resisted thermal inactivation up to 70°C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases.Key words: NKG2D, NK cell, T cell, monoclonal antibody, human IgG1, humanization, phage display, autoimmune disease     

NKG2D and its ligands

NKG2D is an activating immunoreceptor, first recognized on NK cells but subsequently found on γδ T cells, CD8⁺ αβ T cells and macrophages. In NK cells, inhibitory signals are generally dominate over activating signals. However, activating signals mediated through engagement of NKG2D by its ligands on target cells can bypass signals transmitted through inhibitory NK receptors, allowing NKG2D to function as a “master-switch” in determining the activation status of NK cells. NKG2D is important for T cell and NK cell-mediated immunity to viruses and tumours, and has roles in autoimmune disease, allogeneic transplantation, and xenotransplantation. Depending upon the situation, development of strategies to either block or to enhance the interactions between NKG2D and its ligands may have important implications for human health and disease.     

Heterogeneity in phenotype and function of CD8+ and CD4/CD8 double‐negative Natural Killer T cell subsets in sooty mangabeys

Background We have recently reported the presence of CD8⁺ and CD4/8 double‐negative (DN) natural killer T (NKT) lymphocytes in sooty mangabeys. To investigate differences in the two NKT cell subsets, we compared the phenotype and function of sooty mangabey CD8⁺ and DN NKT cells. Methods Flow‐sorted NKT lymphocytes from one SIV‐negative sooty mangabey were subjected to limiting dilution cloning. Invariant NKT clones were characterized by flow cytometry and cytokine ELISA. Results The majority of NKT clones displayed an effector memory phenotype and expressed CXCR3 and NKG2D. While CD8⁺ NKT subsets expressed significantly higher levels of granzyme B and perforin and produced more IFN‐γ, the DN NKT subsets secreted significantly more IL‐4, IL‐13, and IL‐10. Conclusions The Th1 and Th2 cytokine bias of CD8⁺ and DN NKT cells, respectively, indicates the presence of functionally heterogeneous populations of NKT cells in sooty mangabeys.     

Shed NKG2D ligand boosts NK cell immunity

Ligands for natural killer (NK) cell activating receptors can be released from tumor cells and are believed to promote tumor growth by acting as decoys for effector lymphocytes. In a recent paper published in Science, Deng et al. report another scenario in which a shed form of the MULT1 mouse NKG2D ligand boosts NK cell functions.Natural killer (NK) cells are cytolytic and cytokine-producing lymphocytes of the innate immune system that participate in the control of tumor growth and microbial infections¹. NK cell effector activities are tightly controlled by a fine balance of inhibitory and activating signals delivered by surface receptors. Activating receptors can recognize two types of ligands, self-molecules encoded by the host''s own genome whose expression is upregulated upon cellular stress, or exogenous molecules produced by microbes during infection. NKG2D, one of the best characterized activating receptor expressed by NK and T cells, binds to several different ligands in human and mouse². NKG2D ligands are poorly expressed on the vast majority of normal cell surfaces, but are upregulated on tumor and virus-infected cells. In addition, NKG2D ligands can be released by both surface cleavage and exosome excretion. It has been reported that shed ligands can block tumor cell recognition by effector cells by preventing NKG2D interaction with its ligands³. However, several reports do not correlate the presence of soluble ligands with decreased NKG2D expression nor functional activities.Deng et al.⁴ focused their analysis on the NKG2D mouse ligand MULT1, which is commonly overexpressed on primary tumor cells. They first showed that MULT1-transduced fibroblast can cleave MULT1 from the plasma membrane, resulting in a released shed form in the supernatant. Shed MULT1 is of high affinity to NKG2D (∼13 nM) similar to recombinant MULT1. They further reveal the presence of shed MULT1 in the serum of mice developing spontaneous MULT1⁺ tumors. Interestingly, the authors detected a very high concentration of shed MULT1 in the sera of Apoe^−/− mice exhibiting severe atherosclerosis and liver inflammation. Given that these autoimmune injuries observed in this mouse model depend on NKG2D activity⁵, it was unlikely that shed MULT1 exert an inhibitory effect on immunity.Surprisingly, the authors further showed that mouse tumor cells engineered to release a secreted form of MULT1 (secMULT1) similar to the shed MULT1 were rejected when injected into syngenic mice. Tumor rejection is dependent on NK cells as cells grow in NK but not in CD8⁺ T cell-depleted host and requires NKG2D. Importantly, the controlled release of secMULT1 from tumors harboring inducible secMULT1 promotes tumor rejection. To rule out the possibility that tumor cell rejection was due to intrinsic modifications of tumor cells, the author monitored the rejection of a mixture of 9:1 secMULT1⁻: secMULT1⁺ tumor cells and showed an improved antitumoral effect on both secMULT1⁺ and, importantly, secMULT1⁻ tumors. In addition, direct intratumoral injection of recombinant MULT1 promotes tumor rejection. These results suggested that soluble MULT1 mobilizes or activates anti-tumor effector cells. Deng et al. further reported increased frequencies of cytotoxic and IFN-γ-secreting NK cells associated with secMULT1⁺ tumors as compared to control tumor cells. Altogether, these data suggest that a shed NKG2D ligand can promote tumor rejection by boosting NK cell effector functions.Shed MULT1 could crosslink NKG2D and thus activate NK cells. However, shed and secMULT1 are monomeric molecules similar to the recombinant MULT1 which fails to activate NK cells in vitro. Formation of multivalent structures in vivo was not detected. In addition, whereas the transmembrane form of MULT1 can activate NK cells by crosslinking NKG2D and induces NKG2D downregulation, soluble MULT1 upregulates NKG2D on the NK cell surface. This upregulation is probably due do a decreased downregulation of NKG2D surface expression because no increase in NKG2D mRNA or protein was observed. Based on these findings, the authors hypothesized that NKG2D ligands expressed on non-tumor host cell membrane continuously engage NKG2D on NK cells, leading to NKG2D downregulation and NK cell desensitization, whereas soluble MULT1 blocks these interactions to increase NK cell responsiveness (Figure 1). Along this line, NK cells from mutant mice genetically deficient for the NKG2D ligand expressed by tumor-associated myeloid cells are not desensitized.Open in a separate windowFigure 1Tumor-associated cells express NKG2DL which can desensitize NK cells. Tumor shedding of MULT1 delivers soluble MULT1 that outcompetes for NKG2D binding and prevents NK cell desensitization. Boosted NK cell functions lead to improved tumor cell rejection by other activating receptors.The induction of cell desensitization by a frequent or even constant stimulation is a very common mechanism across living objects. Regarding NK cells, another example of tuning via desensitization resides in the impact of the long lasting absence of MHC class I molecules in their environment. Indeed, NK cells are hyporesponsive in a MHC-I-deficient host⁶. There are accumulating data indicating that in the absence of engagement of inhibitory receptors for MHC class I molecules, NK cells get desensitized due to their chronic interaction with endogenous stimulating ligands⁷. Indeed, in the absence of engagement of this inhibitory pathway, NK cell activation would be unleashed⁸. This scenario is supported by a series of in vitro and in vivo experiments in which NK cells are desensitized following chronic exposure to stimulatory molecules expressed at the surface of interacting cells^9,10. Thus, the induction of MHC class I downregulation or NKG2D ligand upregulation boosts NK cell function, whereas the sustained lack of MHC class I or expression of NKG2D ligands impairs NK cell reactivity. This tuning of immune response as a function of the speed of change of the stimuli detected by lymphocytes is at the center of the recently proposed Discontinuity Theory¹¹.Finally, consistent with their findings with secMULT1 but somewhat counter-intuitively, Deng et al. also show that NKG2D receptor deficiency or blockade using anti-NKG2D monoclonal antibodies mimics the effect of soluble MULT1. Indeed, in both conditions, NK cell effector functions are boosted, resulting in improved tumor rejection. Similarly, blocking other NK activating receptors, such as NKp46, may also lead to NK cell desensitization¹². Checkpoint inhibitory receptors are revolutionizing the treatment of cancers by inhibiting the inhibitory receptors. The findings reported by Deng et al. together with earlier results propose alternative strategies of cancer treatment using antibodies that are directed against activating receptors. In the case of NKG2D, the chronic engagement of NK cells with membrane-bound NKG2D ligand affects not only NKG2D-dependent but also NKG2D-independent signaling pathways⁹. The blockade of NKG2D desensitization by antibodies directed against NKG2D should thus also boost NK cell activation via other pathways, such as antibody-dependent cell cytotoxicity. However, the precise identification of the ligand-receptor pair involved in the control of tumors by NK cells will be a limiting factor to these innovative therapeutic approaches. Indeed, antibodies against activating receptors should be designed to boost NK cell reactivity but should not block the recognition of the tumors by NK cells. Finally, as the tuning of NK cell reactivity by soluble NKG2D ligands depends on their affinity for NKG2D, the pre-clinical development of this new class of drug candidates might reveal novel pharmacokinetics and the pharmacodynamics guidelines.     

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94⁺ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94⁺ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL⁺ CD56^dim NK cells, in contrast to the efficient responses by CD56^bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94⁺ KIR2DL^- NK cells may be uniquely beneficial.     

Inhibition of cytokine response to TLR stimulation and alleviation of collagen‐induced arthritis in mice by Schistosoma japonicum peptide SJMHE1

Helminth‐derived products have recently been shown to prevent the development of inflammatory diseases in mouse models. However, most identified immunomodulators from helminthes are mixtures or macromolecules with potentially immunogenic side effects. We previously identified an immunomodulatory peptide called SJMHE1 from the HSP60 protein of Schistosoma japonicum. In this study, we assessed the ability of SJMHE1 to affect murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated by toll‐like receptor (TLR) ligands in vitro and its treatment effect on mice with collagen‐induced arthritis (CIA). We show that SJMHE1 not only modulates the cytokine production of murine macrophage (MΦ) and dendritic cell but also affects cytokine production upon coculturing with allogeneic CD4⁺ T cell. SJMHE1 potently inhibits the cytokine response to TLR ligands lipopolysaccharide (LPS), CpG oligodeoxynucleotides (CpG) or resiquimod (R848) from mouse splenocytes, and human PBMCs stimulated by LPS. Furthermore, SJMHE1 suppressed clinical signs of CIA in mice and blocked joint erosion progression. This effect was mediated by downregulation of key cytokines involved in the pathogenesis of CIA, such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6, IL‐17, and IL‐22 and up‐regulation of the inhibitory cytokine IL‐10, Tgf‐β1 mRNA, and CD4⁺CD25⁺Foxp3⁺ Tregs. This study provides new evidence that the peptide from S. japonicum, which is the ‘safe’ selective generation of small molecule peptide that has evolved during host–parasite interactions, is of great value in the search for novel anti‐inflammatory agents and therapeutic targets for autoimmune diseases.     

Alteration of inhibitory and activating natural killer cell receptor expression on T cells in human immunodeficiency virus-infected Chinese

T cell expression of NKRs can trigger or inhibit cell‐mediated cytotoxicity. However, few studies on T lymphocyte NKR expression in HIV infection exist. Here, we examined the expression patterns of NKG2D, NKG2A, and KIR3DL1 on CD8⁺ and CD3⁺CD8^? cells by multicolor flow cytometry in groups of patients with HIV, AIDS or HAART‐treated AIDS, as well as HIV‐negative normal controls. Individual analysis of KIR3DL1 on CD3⁺CD8⁺ or CD3⁺CD8^? cells revealed no significant differences among any of the groups (P > 0.05). In contrast, the percentage of NKG2A⁺NKG2D^?CD8⁺ T cells was higher in the AIDS group than in the HIV‐negative normal control group (P < 0.01). Meanwhile, the prevalence of NKG2D⁺NKG2A^?CD8⁺T cells was lower in the AIDS group than in HIV‐negative normal controls (P < 0.001). Similar results were also observed for the percentage of NKG2A⁺NKG2D^? on CD3⁺CD8^?cells. However, in contrast to CD8⁺ T cells, the frequencies of NKG2D⁺NKG2A^? on CD3⁺CD8^? cells were higher in AIDS and HIV patients than in HIV‐negative normal controls (P < 0.01, P < 0.05, respectively). The percentage of NKG2A⁺NKG2D^?CD8⁺ T cells was negatively correlated with CD4⁺ T cell counts (r=?0.499, P < 0.01), while the percentage of NKG2D⁺NKG2A^?CD8⁺ T cells was positively correlated with CD4⁺ T cell counts (r= 0.494, P < 0.01). The percentage of NKG2D⁺NKG2A^?CD3⁺CD8^? T cells was also positively correlated with viral load (r= 0.527, P < 0.01) and negatively correlated with CD4⁺ T cell counts (r=?0.397, P < 0.05). Finally, HAART treatment reversed the changes in NKR expression caused by HIV infection. These results indicate that the expression of NKRs on T cells may be correlated with HIV disease progression.     

Regulation of arginase I activity and expression by both PD-1 and CTLA-4 on the myeloid-derived suppressor cells

An elevated number of Gr-1⁺CD11b⁺ myeloid-derived suppression cells (MDSCs) has been described in mice and human bearing tumor and associated with immune suppression. Arginase I production by MDSCs in the tumor environment may be a central mechanism for immunosuppression and tumor evasion. In this study and before, we found that Gr-1⁺CD11b⁺ MDSCs from ascites and spleen of mice bearing ovarian 18D carcinoma express a high level of PD-1, CTLA-4, B7-H1 and CD80 while other co-stimulatory molecules, namely CD40, B7-DC and CD86 are not detected. Further studies showed that PD-1 and CTLA-4 on the Gr-1⁺CD11b⁺ MDSCs regulated the activity and expression of arginase I. The blockage and silencing of PD-1, CTLA-4 or both PD-1 and CTLA4 molecules could significantly reduce arginase I activity and expression induced with tumor-associated factor. Similar results were also observed while their ligands B7-H1 and/or CD80 were blocked or silenced. Furthermore, CD80 deficiency also decreased the arginase I expression and activity. Antibody blockade or silencing of PD-1, CTLA-4 or both reduced the suppressive potential of PD-1+CTLA-4+MDSCs. Blockade of PD-1, CTLA-4 or both also slowed tumor growth and improved the survival rate of tumor-bearing mice. Thus, there may exist a coinhibitory and costimulatory molecules-based immuno-regulating wet among MDSCs. This research was supported by Nankai University grant, NSFC grant “30771967”, “985” grant,The Ministry of Science and Technology grant “2006AA020502”“06C26211200695”, Tianjin Grant “07JCZDJC03300” and “06ZHCXSH04800”.     

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.     

Alteration of inhibitory and activating NK cell receptor expression on NK cells in HIV-infected Chinese

Natural killer (NK) cell function, based on the expression of activating and inhibitory natural killer receptors (NKRs), may become abnormal during human immunodeficiency virus (HIV) infection. In this study, we investigated changes in receptor expression with individual and combinational analysis on NK cell subsets in HIV-infected Chinese. The results showed that natural killer group 2 member D (NKG2D) expression on total NK cells decreased significantly in HIV infection, while the expressions of natural killer group 2 member A (NKG2A) and killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail 1 (KIR3DL1) on total NK cells were not significantly different between any of the groups including HIV-positive treatment-naïve group, AIDS treatment-naïve group, HAART-treatment AIDS group and HIV-negative control group. Individual analysis of NKG2A⁺ and KIR3DL1⁺ cells revealed no significant differences in expression in any NK cell subsets between any of the groups, but the combinational analysis of NKG2D⁻NKG2A⁺, and NKG2D⁻KIR3DL1⁺ on the NK CD56^dim cell subset in the AIDS group were increased compared to the HIV-negative control group. On the contrary, NKG2D⁻NKG2A⁺ expression on the CD56^bright subset decreased in the AIDS group compared to the control group. Highly active antiretroviral therapy (HAART) treatment almost completely restored the levels of these receptor expressions. The results indicate that the distinct alteration of activating and inhibitory NKR expression on NK cells and its subsets occurred during HIV progression. Moreover, the imbalanced change of activating and inhibitory NKRs on NK cells and its subsets may explain the impaired NK cell immunity in HIV infected individuals.     

High‐dose wogonin exacerbates DSS‐induced colitis by up‐regulating effector T cell function and inhibiting Treg cell

Wogonin exerts anti‐tumour activities via multiple mechanisms. We have identified that high‐dose wogonin (50 or 100 mg/kg) could inhibit the growth of transplanted tumours by directly inducing tumour apoptosis and promoting DC, T and NK cell recruitment into tumour tissues to enhance immune surveillance. However, wogonin (20–50 μM) ex vivo prevents inflammation by inhibiting NF‐κB and Erk signalling of macrophages and epithelial cells. It is elusive whether high‐dose wogonin promotes or prevents inflammation. To investigate the effects of high‐dose wogonin on murine colitis induced by dextran sodium sulphate (DSS), mice were co‐treated with DSS and various doses of wogonin. Intraperitoneal administration of wogonin (100 mg/kg) exacerbated DSS‐induced murine colitis. More CD4⁺ CD44⁺ and CD8⁺ CD44⁺ cells were located in the inflamed colons in the wogonin (100 mg/kg) treatment group than in the other groups. Frequencies of CD4⁺ CD25⁺ CD127^? and CD4⁺ CD25⁺ Foxp3⁺ cells in the colons and spleen respectively, were reduced by wogonin treatment. Ex vivo stimulations with high‐dose wogonin (50–100 μg/ml equivalent to 176–352 μM) could synergize with IL‐2 to promote the functions of CD4⁺ and CD8⁺ cells. However, regulatory T cell induction was inhibited. Wogonin stimulated the activation of NF‐κB and Erk but down‐regulated STAT3 phosphorylation in the CD4⁺ T cells. Wogonin down‐regulated Erk and STAT3‐Y705 phosphorylation in the regulatory T cells but promoted NF‐κB and STAT3‐S727 activation. Our study demonstrated that high‐dose wogonin treatments would enhance immune activity by stimulating the effector T cells and by down‐regulating regulatory T cells.     

CD8+ T cells exhaustion induced by myeloid‐derived suppressor cells in myelodysplastic syndromes patients might be through TIM3/Gal‐9 pathway

CD8⁺ T cells play a central role in antitumour immunity, which often exhibit ‘exhaustion’ in the setting of malignancy and chronic viral infection due to T cell immunoglobulin and mucin domain 3 (TIM3) and myeloid‐derived suppressor cells (MDSCs). Our team previously found that overactive MDSCs and exhausted TIM3⁺CD8⁺ T cells were observed in myelodysplastic syndromes (MDS) patients. However, it is not obvious whether MDSCs suppress CD8⁺ T cells through TIM3/Gal‐9 pathway. Here, Gal‐9, as the ligand of TIM3, was overexpressed in MDSCs. The levels of Gal‐9 in bone marrow supernatants, serum and culture supernatants of MDSCs from MDS patients were elevated. CD8⁺ T cells from MDS or normal controls produced less perforin and granzyme B and exhibited increased early apoptosis after co‐culture with MDSCs from MDS. Meanwhile, the cytokines produced by CD8⁺ T cells could be partially restored by TIM3/Gal‐9 pathway inhibitors. Furthermore, CD8⁺ T cells produced less perforin and granzyme B after co‐culture with excess exogenous Gal‐9, and the function of CD8⁺ T cells was similarly restored by TIM3/Gal‐9 pathway inhibitors. Expression of Notch1, EOMES (associated with perforin and granzyme B secretion), p‐mTOR and p‐AKT (associated with cell proliferation) was decreased in CD8⁺ T cells from MDS after co‐culture with excess exogenous Gal‐9. These suggested that MDSCs might be the donor of Gal‐9, and TIM3/Gal‐9 pathway might be involved in CD8⁺ T cells exhaustion in MDS, and that TIM3/Gal‐9 pathway inhibitor might be the promising candidate for target therapy of MDS in the future.