Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation.

BACKGROUND: The conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. METHODS: An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-gamma-secreting cells in HIFU-treated mice. RESULTS: HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ. CONCLUSION: Optimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.

On the Origin of Natural Antibody

Natural IgM (nIgM) is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. nIgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. However, the cells that maintain high titers of nIgM in the circulation had not yet been identified. Several studies have linked serum nIgM with the presence of fetal-lineage B cells, and others have detected IgM secretion directly by B1a cells in various tissues. Nevertheless, a substantial contribution of undifferentiated B1 cells to nIgM titers is doubtful, as the ability to produce large quantities of antibody (Ab) is a function of the phenotype and morphology of differentiated plasma cells (PCs). No direct evidence exists to support the claim that a B1-cell population directly produces the bulk of circulating nIgM. The source of nIgM thus remained uncertain and unstudied.

In the first part of this study, I identified the primary source of nIgM. Using enzyme-linked immunosorbent spot (ELISPOT) assay, I determined that the majority of IgM Ab-secreting cells (ASCs) in naïve mice reside in the bone marrow (BM). Flow cytometric analysis of BM cells stained for intracellular IgM revealed that nIgM ASCs express IgM and the PC marker CD138 on their surface, but not the B1a cell marker CD5. By spinning these cells onto slides and staining them, following isolation by fluorescence-activated cell sorting (FACS), I found that they exhibit the typical morphological characteristics of terminally differentiated PCs. Transfer experiments demonstrated that BM nIgM PCs arise from a progenitor in the peritoneal cavity (PerC), but not isolated PerC B1a, B1b, or B2 cells. Immunoglobulin (Ig) gene sequence analysis and examination of B1-8i mice, which carry an Ig knockin that prohibits fetal B-cell development, indicated that nIgM PCs differentiate from fetal-lineage B cells. BrdU uptake experiments showed that the nIgM ASC compartment contains a substantial fraction of long-lived plasma cells (LLPCs). Finally, I demonstrated that nIgM PCs occupy a survival niche distinct from that used by IgG PCs.

In the second part of this dissertation, I characterized the unique survival niche of nIgM LLPCs, which maintain constitutive high titers of nIgM in the serum. By using genetically deficient or Ab-depleted mice, I found that neither T cells, type 2 innate lymphoid cells, nor mast cells, the three major hematopoietic producers of IL-5, were required for nIgM PC survival in the BM. However, IgM PCs associate strongly with IL-5-expressing BM stromal cells, which support their survival in vitro when stimulated. In vivo neutralization of IL-5 revealed that, like individual survival factors for IgG PCs, IL-5 is not the sole supporter of IgM PCs, but is likely one of several redundant molecules that together ensure uninterrupted signaling. Thus, the long-lived nIgM PC niche is not composed of hematopoietic sources of IL-5, but a stromal cell microenvironment that provides multiple redundant survival signals.

In the final part of my study, I identified and characterized the precursor of nIgM PCs, which I found in the first project to be resident in the PerC, but not a B1a, B1b, or B2 cell. By transferring PerC cells sorted based on expression of CD19, CD5, and CD11b, I found that only the CD19+CD5+CD11b- population contained cells capable of differentiating into nIgM PCs. Transfer of decreasing numbers of unfractionated PerC cells into Rag1 knockouts revealed an order-of-magnitude drop in the rate of serum IgM reconstitution between stochastically sampled pools of 106 and 3x105 PerC cells, suggesting that the CD19+CD5+CD11b- compartment comprises two cell types, and that interaction between the two necessary for nIgM-PC differentiation. By transferring neonatal liver, I determined that the early hematopoietic environment is required for nIgM PC precursors to develop. Using mice carrying a mutation that disturbs cKit expression, I also found that cKit appears to be required at a critical point near birth for the proper development of nIgM PC precursors.

The collective results of these studies demonstrate that nIgM is the product of BM-resident PCs, which differentiate from a PerC B cell precursor distinct from B1a cells, and survive long-term in a unique survival niche created by stromal cells. My work creates a new paradigm by which to understand nIgM, B1 cell, and PC biology.

Acute administration of unacylated ghrelin has no effect on Basal or stimulated insulin secretion in healthy humans.

Unacylated ghrelin (UAG) is the predominant ghrelin isoform in the circulation. Despite its inability to activate the classical ghrelin receptor, preclinical studies suggest that UAG may promote β-cell function. We hypothesized that UAG would oppose the effects of acylated ghrelin (AG) on insulin secretion and glucose tolerance. AG (1 µg/kg/h), UAG (4 µg/kg/h), combined AG+UAG, or saline were infused to 17 healthy subjects (9 men and 8 women) on four occasions in randomized order. Ghrelin was infused for 30 min to achieve steady-state levels and continued through a 3-h intravenous glucose tolerance test. The acute insulin response to glucose (AIRg), insulin sensitivity index (SI), disposition index (DI), and intravenous glucose tolerance (kg) were compared for each subject during the four infusions. AG infusion raised fasting glucose levels but had no effect on fasting plasma insulin. Compared with the saline control, AG and AG+UAG both decreased AIRg, but UAG alone had no effect. SI did not differ among the treatments. AG, but not UAG, reduced DI and kg and increased plasma growth hormone. UAG did not alter growth hormone, cortisol, glucagon, or free fatty acid levels. UAG selectively decreased glucose and fructose consumption compared with the other treatments. In contrast to previous reports, acute administration of UAG does not have independent effects on glucose tolerance or β-cell function and neither augments nor antagonizes the effects of AG.

Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.

The peptide tyrosine tyrosine (PYY) is produced and secreted from L cells of the gastrointestinal mucosa. To study the anatomy and function of PYY-secreting L cells, we developed a transgenic PYY-green fluorescent protein mouse model. PYY-containing cells exhibited green fluorescence under UV light and were immunoreactive to antibodies against PYY and GLP-1 (glucagon-like peptide-1, an incretin hormone also secreted by L cells). PYY-GFP cells from 15 μm thick sections were imaged using confocal laser scanning microscopy and three-dimensionally (3D) reconstructed. Results revealed unique details of the anatomical differences between ileal and colonic PYY-GFP cells. In ileal villi, the apical portion of PYY cells makes minimal contact with the lumen of the gut. Long pseudopod-like basal processes extend from these cells and form an interface between the mucosal epithelium and the lamina propria. Some basal processes are up to 50 μm in length. Multiple processes can be seen protruding from one cell and these often have a terminus resembling a synapse that appears to interact with neighboring cells. In colonic crypts, PYY-GFP cells adopt a spindle-like shape and weave in between epithelial cells, while maintaining contact with the lumen and lamina propria. In both tissues, cytoplasmic granules containing the hormones PYY and GLP-1 are confined to the base of the cell, often filling the basal process. The anatomical arrangement of these structures suggests a dual function as a dock for receptors to survey absorbed nutrients and as a launching platform for hormone secretion in a paracrine fashion.

Exhausted CD8 T cells downregulate the IL-18 receptor and become unresponsive to inflammatory cytokines and bacterial co-infections.

During many chronic infections virus-specific CD8 T cells succumb to exhaustion as they lose their ability to respond to antigenic activation. Combinations of IL-12, IL-18, and IL-21 have been shown to induce the antigen-independent production of interferon (IFN)-γ by effector and memory CD8 T cells. In this study we investigated whether exhausted CD8 T cells are sensitive to activation by these cytokines. We show that effector and memory, but not exhausted, CD8 T cells produce IFN-γ and upregulate CD25 following exposure to certain combinations of IL-12, IL-18, and IL-21. The unresponsiveness of exhausted CD8 T cells is associated with downregulation of the IL-18-receptor-α (IL-18Rα). Although IL-18Rα expression is connected with the ability of memory CD8 T cells to self-renew and efflux rhodamine 123, the IL-18Rα(lo) exhausted cells remained capable of secreting this dye. To further evaluate the consequences of IL-18Rα downregulation, we tracked the fate of IL-18Rα-deficient CD8 T cells in chronically infected mixed bone marrow chimeras and discovered that IL-18Rα affects the initial but not later phases of the response. The antigen-independent responsiveness of exhausted CD8 T cells was also investigated following co-infection with Listeria monocytogenes, which induces the expression of IL-12 and IL-18. Although IL-18Rα(hi) memory cells upregulated CD25 and produced IFN-γ, the IL-18Rα(lo) exhausted cells failed to respond. Collectively, these findings indicate that as exhausted T cells adjust to the chronically infected environment, they lose their susceptibility to antigen-independent activation by cytokines, which compromises their ability to detect bacterial co-infections.