Regulation of development of CD56 bright CD11c + NK-like cells with helper function by IL-18.

Human γδ T cells augment host defense against tumors and infections, and might have a therapeutic potential in immunotherapy. However, mechanism of γδ T cell proliferation is unclear, and therefore it is difficult to prepare sufficient numbers of γδ T cells for clinical immunotherapy. Recently, natural killer (NK)-like CD56(bright)CD11c(+) cells were shown to promote the proliferation of γδ T cells in an IL-18-dependent manner. In this study, we demonstrated that the NK-like CD56(bright)CD11c(+) cells could directly interact with γδ T cells to promote their sustained expansion, while conventional dendritic cells (DCs), IFN-α-induced DCs, plasmacytoid DCs or monocytes did not. We also examined the cellular mechanism underlying the regulation of CD56(bright)CD11c(+) cells. CD14(+) monocytes pre-incubated with IL-2/IL-18 formed intensive interactions with CD56(int)CD11c(+) cells to promote their differentiation to CD56(bright)CD11c(+) cells with helper function. The development of CD56(bright)CD11c(+) cells was suppressed in an IFN-α dependent manner. These results indicate that CD14(+) monocytes pretreated with IL-2/IL-18, but neither DCs nor monocytes, play a determining role on the development and proliferation of CD56(bright)CD11c(+) cells, which in turn modulate the expansion of γδ T cells. CD56(bright)CD11c(+) NK-like cells may be a novel target for immunotherapy utilizing γδ T cells, by overcoming the limitation of γδ T cells proliferation

Development, change of phenotype and function of CD56^brightCD11c⁺ cells induced by IL-18.

<p>(A) Time course of the development of CD56^brightCD11c⁺ cells. PBMCs (1×10⁵ cells/0.5 ml/well) were stimulated with ZOL/IL-2/IL-18. The absolute numbers of CD56⁺ or CD56^brightCD11c⁺ cells (white bar), γδ T cells (black bar) and αβ T cells (gray bar) were determined by flow cytometry and trypan blue dye exclusion test at day 0, day 3 (left), and day 7 (right). Data show mean ± SD (n = 10), **p<0.01. (B) Proliferation of CD56⁺ or CD56^brightCD11c⁺ cells (red line), γδ T cells (black line), and αβ T cells (gray line). Data show mean ± SD (n = 10), **p<0.01. (C) Requirement of CD56^brightCD11c⁺ cells for maximal sustained proliferation of γδ T cells. PBMCs were pre-stimulated with ZOL/IL-2/IL-18, and harvested on day 7. The proliferating cells were divided into 2 groups: one group was incubated with anti-CD56 antibody-conjugated beads and CD56⁺ cells were selectively removed. The other group was incubated with mouse IgG1-conjugated beads and used as a control. Both groups were re-incubated with ZOL/IL-2/IL-18 for another 14 days. Data show mean ± SD (n = 5), **p<0.01. (D) Development of CD56^brightCD11c⁺cells and gradual reduced expression of CD11c. The number and CD11c expression of proliferated cells were analyzed by flow cytometry during the culture of CD3⁺ T cell-depleted PBMCs. (Grey shadow: isotype control; blue: CD56^intCD11c⁺cells on day 0; red: CD56^brightCD11c⁺cells on day 7; thin black line: day 14; and bold black line: day 21). Data show mean ± SD (n = 5). A histogram shown is a representative of five independent experiments. (E) Comparison among CD56^brightCD11c⁺ cells, monocytes, and several subsets of DCs in helper activity for γδ T cells proliferation. Freshly isolated γδ T cells(5×104/well) were labeled with CFSE and co-cultured for 7 days with fresh CD14⁺ monocytes, IFN-α-DCs, IL-4-DCs, CD56^brightCD11c⁺, or pDCs, at a ratio of 1∶1 in the presence of ZOL/IL-2/IL-18. Then, proliferative responses of γδ T cells were analyzed based on flow cytometry and trypan blue dye exclusion test. R1: γδ T cells labeled with CFSE undergoing cell division; R2: Unlabeled CD56^brightCD11c⁺ cells. Data show mean ± SD (n = 5), **p<0.01. The result is representative of five independent experiments. (F) Differential cytotoxicity of CD56^brightCD11c⁺ cells against normal osteoblast cells (NHOst) and tumor cells (MG-63). The cytotoxicity was assessed using standard propidium iodide staining. Dot plots shown are representative of three independent experiments.</p

Putative model for the regulation of the development and expansion of CD56^brightCD11c⁺ cells and γδ T cells.

<p>In response to ZOL, CD14⁺ monocytes stimulate γδ T cells in a TCR-dependent manner. Concomitantly, CD14⁺ monocytes induce the IL-2/IL-18-mediated generation of CD56^brightCD11c⁺ cells from their putative precursor CD56^intCD11c⁺ cells. IFN-α inhibits this process possibly through the production of IFN-α-DCs. The resulting CD56^brightCD11c⁺ cells initiate and promote the expansion of γδ T cells for several days and gradually lose their helper function as they lose CD11c expression.</p

Negative regulation of CD56^brightCD11c⁺ cell development by IFN-α.

<p>(A) Inhibition of γδ T cell proliferation by IFN-α in PBMC cultures. PBMCs were stimulated with ZOL/IL-2 for 10 days in presence of various doses of IFN-α. Data show mean ± SD (n = 5), **p<0.01. (B) Attenuation by IFN-α of IL-18-mediated γδ T cell expansion in PBMC cultures with ZOL/IL-2. Data show mean ± SD (n = 4), **p<0.01. (C) Abrogation by IFN-α of the development and proliferation of CD56^brightCD11c⁺ cells in cultures of CD3⁺ T cells-depleted PBMCs. Data show mean ± SD (n = 4), **p<0.01. (D) Inhibition of cell aggregation by IFN-α in cultures of CD56^intCD11c⁺ cells and CD14⁺ monocytes, in the absence and presence of freshly isolated γδ T cells. CD14⁺ monocytes were pretreated with IL-2/IL-18 for 3 days, with or without IFN-α then CD56^intCD11c⁺ cells were added into the culture (upper panels). Next, freshly isolated γδ T cells were added and cellular clusters were observed by microscope (lower panels). The cell aggregates image is representative of three independent experiments. (E) Proliferation of γδ T cells in cultures containing mature CD56^brightCD11c⁺ cells even in the presence of IFN-α. Freshly isolated γδ T cells and mature CD56^brightCD11c⁺ cells were stimulated with ZOL/IL-2, with or without further addition of IFN-α since day 7 onwards and were continuously incubated. The number of proliferating cells was assayed after another 7 days' culture. Data show mean ± SD (n = 5).</p

Interaction between γδ T cells and CD56^brightCD11c⁺cells.

<p>(A) Cell aggregates observed in culture of γδ T cells mixed with CD56^brightCD11c⁺ cells without pulse (left), pulsed with ZOL (center), and further incubated with ZOL (right) (upper panels) and confocal microscopic observation of the culture of γδ T cells (green) and CD56^brightCD11c⁺ cells (red) in the presence of ZOL (lower panels). Data shown are representative of three independent experiments. (B) Flow cytometric analysis of γδ T cells incubated with CD56^brightCD11c+ cells (upper panels) and the numbers of γδ T cells after expansion for 7 days (lower panel). Dot plots shown are representative of three independent experiments, and data of cell counts show mean ± SD (n = 5), *p<0.05, **p<0.01. (C) There was no sustained proliferation of freshly isolated γδ T cells in the absence of accessory cells even after stimulation with IL-2, IL-2/ZOL, IL-2/IL-18/ZOL, or IL-2/2M3B1PP for 7 days. Data show the mean ± SD (n = 5). (D) Expression of co-stimulatory molecules and chemokine receptors on γδ T cells after culture with ZOL, IL-2, or ZOL/IL-2 for day 7. The result of flow cytometric analysis is representative of three independent experiments. (E) Induction of CCL21 by IL-18 in CD56^brightCD11c⁺cells. CCL21 concentration in the culture supernatant at 7 days was measured by ELISA. Data show mean ± SD (n = 5), *p<0.05.</p

A prospective investigation of impacts of comorbid attention deficit hyperactivity disorder (ADHD) on clinical features and long-term treatment response in adult patients with obsessive-compulsive disorder (OCD)

Background: A close association between obsessive-compulsive disorder (OCD) and attention-deficit/hyperactivity disorder (ADHD) in children and adolescents has been investigated in previous studies. However, few studies examined the relationship between lifetime comorbidity of ADHD and OCD in adults. Therefore, we sought to investigate the clinical and psychopathological features related to comorbid ADHD in Japanese adult patients with OCD. Methods: We assessed lifetime comorbidity of ADHD in 93 adult Japanese patients with OCD. Additionally, we used the Japanese version of Conners' Adult ADHD Rating Scales to assess the characteristics and severity of ADHD in each participant. According to the results, we excluded OCD patients that did not have ADHD but who exhibited elevated levels of ADHD traits. We compared OCD patients with ADHD (ADHD+ group) and those without ADHD or its trait (ADHD− group) in terms of background profiles and clinical features, such as OCD symptomatology and psychometric test results. Additionally, the 6-month treatment outcome was compared prospectively between groups. Results: Of the 93 OCD participants, the prevalence of lifetime comorbidity of ADHD was estimated as 16.1%. Compared with the ADHD− group, participants in the ADHD+ group had an earlier age of onset of OCD, higher frequencies of hoarding symptoms, higher levels of depressive and anxiety symptoms and lower quality of life, more elevated levels of impulsivity, and higher rates of substance or behavioral addiction and major depression. Finally, the mean improvement rate on the Yale-Brown Obsessive Compulsive Scale after 6 months of standardized OCD treatment in the ADHD+ group (16.1%) was significantly lower than that in the ADHD− group (44.6%). Conclusion: The lifetime comorbidity of ADHD is likely to exert a significant effect on clinical features and treatment outcome in adult patients with OCD. It is important to consider that underlying ADHD pathology may function as a facilitator for increased severity of global clinical features and treatment refractory conditions in OCD patients. Further studies are required to examine treatment strategies for such patients

Phenotypic and functional analyses of CD14⁺ monocytes involved in the generation of CD56^brightCD11c⁺ cells.

<p>(A) Effect of IL-2/IL-18 on CD14⁺ monocytes proliferation. Data show mean ± SD (n = 4). (B) Analysis of IL-18 receptors and LFA-1 expressed by fresh CD14⁺ monocytes by flow cytometry. A representative result of three independent experiments is shown. (C) Flow cytometric analysis of CD14⁺ monocytes stimulated with IL-2/IL-18 after 24h culture. A representative result of three independent experiments is shown. (D) Analysis of cellular interactions between enriched CD56⁺ cells and purified CD14⁺ monocytes. Purified CD14⁺ monocytes were placed in the lower chambers and CD56⁺ cells containing CD56^intCD11c⁺ cells in the upper chambers. After incubation for 5 days in the presence of IL-2/IL-18, the number of cells in lower chambers was counted and the frequency of cells in the lower chambers was analyzed by flow cytometry. Data show mean ± SD (n = 4), **p<0.01.</p