Differentiation of cultured hair follicle neural crest stem cells into functional melanocytes.

Many autologous melanocytes are required for surgical treatment of depigmentation diseases such as vitiligo. However, primary cultured melanocytes have a limited number of in vitro passages. The production of functional epidermal melanocytes from stem cells provides an unprecedented source of cell therapy for vitiligo. This study explores the clinical application of melanocytes induced by hair follicle neural crest stem cells (HFNCSCs). This study established an in vitro differentiation model of HFNCSCs into melanocytes. Results demonstrate that most differentiated melanocytes expressed the proteins C-KIT, MITF, S-100B, TYRP1, TYRP2, and tyrosinase. The HFNCSC-derived melanocytes were successfully transplanted onto the dorsal skin of mice and survived in the local tissues, expressing marker protein of melanocytes. In conclusion, HFNCSCs in mice can be induced to differentiate into melanocytes under specific conditions. These induced melanocytes exhibit the potential to facilitate repigmentation in the lesion areas of vitiligo-affected mice, suggesting a promising avenue for therapeutic intervention.

Neutrophil-activating therapy for the treatment of cancer.

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

Genome-Wide Analysis of Ribosomal Protein GhRPS6 and Its Role in Cotton Verticillium Wilt Resistance.

Verticillium wilt is threatening the world's cotton production. The pathogenic fungus Verticillium dahliae can survive in the soil in the form of microsclerotia for a long time, colonize through the root of cotton, and invade into vascular bundles, causing yellowing and wilting of cotton leaves, and in serious cases, leading to plant death. Breeding resistant varieties is the most economical and effective method to control Verticillium wilt. In previous studies, proteomic analysis was carried out on different cotton varieties inoculated with V. dahliae strain Vd080. It was found that GhRPS6 was phosphorylated after inoculation, and the phosphorylation level in resistant cultivars was 1.5 times than that in susceptible cultivars. In this study, knockdown of GhRPS6 expression results in the reduction of SA and JA content, and suppresses a series of defensive response, enhancing cotton plants susceptibility to V. dahliae. Overexpression in Arabidopsis thaliana transgenic plants was found to be more resistant to V. dahliae. Further, serines at 237 and 240 were mutated to phenylalanine, respectively and jointly. The transgenic Arabidopsis plants demonstrated that seri-237 compromised the plant resistance to V. dahliae. Subcellular localization in Nicotiana benthamiana showed that GhRPS6 was localized in the nucleus. Additionally, the pathogen inoculation and phosphorylation site mutation did not change its localization. These results indicate that GhRPS6 is a potential molecular target for improving resistance to Verticillium wilt in cotton. This lays a foundation for breeding disease-resistant varieties.

Mechanical Stiffness Controls Dendritic Cell Metabolism and Function.

Stiffness in the tissue microenvironment changes in most diseases and immunological conditions, but its direct influence on the immune system is poorly understood. Here, we show that static tension impacts immune cell function, maturation, and metabolism. Bone-marrow-derived and/or splenic dendritic cells (DCs) grown in vitro at physiological resting stiffness have reduced proliferation, activation, and cytokine production compared with cells grown under higher stiffness, mimicking fibro-inflammatory disease. Consistently, DCs grown under higher stiffness show increased activation and flux of major glucose metabolic pathways. In DC models of autoimmune diabetes and tumor immunotherapy, tension primes DCs to elicit an adaptive immune response. Mechanistic workup identifies the Hippo-signaling molecule, TAZ, as well as Ca2+-related ion channels, including potentially PIEZO1, as important effectors impacting DC metabolism and function under tension. Tension also directs the phenotypes of monocyte-derived DCs in humans. Thus, mechanical stiffness is a critical environmental cue of DCs and innate immunity.

Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells.

Dendritic cells (DCs) are powerful antigen presenting cells, derived from bone marrow progenitors (cDCs) and monocytes (moDCs), that can shape the immune response by priming either proinflammatory or tolerogenic immune effector cells. The cellular mechanisms responsible for the generation of DCs that will prime a proinflammatory or tolerogenic response are poorly understood. Here we describe a novel mechanism by which tolerogenic DCs are formed from monocytes. When human monocytes were cultured with CD4+FoxP3+ natural regulatory T cells (Tregs) and T helper cells (Th) from healthy donor blood, they differentiated into regulatory DCs (DC Reg ), capable of generating induced Tregs from naïve T cells. DC Reg exhibited morphology, surface phenotype, cytokine secretion, and transcriptome that were distinct from other moDCs including those derived from monocytes cultured with Th or with GM-CSF/IL-4, as well as macrophages (MΦ). Direct cell contact between monocytes, Tregs and Th, along with Treg-derived CTLA-4, IL-10 and TGF-ß, was required for the phenotypic differentiation of DC Reg , although only IL-10 was required for imprinting the Treg-inducing capacity of DC Reg . High ratios of Treg:Th, along with monocytes and DC Reg similar in function and phenotype to those induced in vitro, were present in situ in human colorectal cancer specimens. Thus, through the combined actions of Tregs and Th, monocytes differentiate into DCs with regulatory properties, forming a positive feedback loop to reinforce Treg initiated immune regulation. This mechanism may contribute to immune tolerance in tissues such as tumors, which contain an abundance of Tregs, Th and monocytes.

Tolerogenic interactions between CD8+ dendritic cells and NKT cells prevent rejection of bone marrow and organ grafts.

The combination of total lymphoid irradiation and anti-T-cell antibodies safely induces immune tolerance to combined hematopoietic cell and organ allografts in humans. Our mouse model required host natural killer T (NKT) cells to induce tolerance. Because NKT cells normally depend on signals from CD8+ dendritic cells (DCs) for their activation, we used the mouse model to test the hypothesis that, after lymphoid irradiation, host CD8+ DCs play a requisite role in tolerance induction through interactions with NKT cells. Selective deficiency of either CD8+ DCs or NKT cells abrogated chimerism and organ graft acceptance. After radiation, the CD8+ DCs increased expression of surface molecules required for NKT and apoptotic cell interactions and developed suppressive immune functions, including production of indoleamine 2,3-deoxygenase. Injection of naive mice with apoptotic spleen cells generated by irradiation led to DC changes similar to those induced by lymphoid radiation, suggesting that apoptotic body ingestion by CD8+ DCs initiates tolerance induction. Tolerogenic CD8+ DCs induced the development of tolerogenic NKT cells with a marked T helper 2 cell bias that, in turn, regulated the differentiation of the DCs and suppressed rejection of the transplants. Thus, reciprocal interactions between CD8+ DCs and invariant NKT cells are required for tolerance induction in this system that was translated into a successful clinical protocol.

Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity.

BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain-containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity.

Neonatal lymph node stromal cells drive myelodendritic lineage cells into a distinct population of CX3CR1+CD11b+F4/80+ regulatory macrophages in mice.

Beyond providing a scaffold for immune cells, recent studies indicate that lymph node stromal cells provide potent regulatory capacities that affect the quality of adaptive immune responses. In this study, we provide evidence that neonatal lymph node stromal cells (nnLNSCs) consistently promote the differentiation of macrophage dendritic cell progenitors as well as mature and immature dendritic cells into a distinct population of CX3CR1(+) CD11b(+)F4/80(+) regulatory macrophages (regMΦ). These cells possess remarkably low levels of T cell costimulatory molecules as well as MHC class II molecules. regMΦ do not interfere with early T-cell activation but, via nitric oxide secretion, efficiently suppress T-cell proliferation. Furthermore, CD4(+) T cells proliferating in the presence of regMΦ gain immunosuppressive capacity and MΦ isolated from day 3 nnLNs are T-cell immunosuppressive. Adoptive transfer of antigen-loaded regMΦ induce a profound antigen-specific immune suppression in vivo. Together our data show that nnLNSCs skew the differentiation of dendritic cells and their progenitors toward regMΦ, thus revealing a novel mechanism for local immune regulation.

Identification and expression analysis of alternatively spliced isoforms of human interleukin-23 receptor gene in normal lymphoid cells and selected tumor cells.

Interleukin 23 (IL-23) is a new member of the IL-12 family that plays a critical role in promoting the proliferation of memory T helper 1 cells. The heterodimerized IL-23 receptor is composed of a shared IL-12 receptor beta 1 (IL-12Rbeta1) and an IL-12Rbeta2-related molecule called IL-23R. The standard form of IL-23R is encoded by at least 12 exons. Here, we demonstrate that at least six spliced isoforms of IL-23R (IL-23R1 to 6) can be generated through alternative splicing. The splicing strategies for the IL-23R gene are complicated and most often result in the deletion of exon 7 and/or exon 10. Translation prediction revealed that these spliced variants result in either premature termination to give rise to a diverse form of receptor ectodomain, or a frameshift to generate various lengths of the IL-23R endodomain. Differential expressions of IL-23R spliced variants are observed in natural killer and CD3+ CD4+ T cells. The expressions of these spliced variants are also prevalently and complicatedly regulated in tumor cell lines. Interestingly, only IL-23R2 and/or IL-23R4 variants are predominantly detected in certain human lung carcinomas, but not in their resected normal margin tissues. Thus, our results indicate that the regulation of alternative splicing on the IL-23R gene is complicated, and the preferential expression of certain IL-23R spliced variants may be a contributive factor to the pathogenesis of certain cancers.