Optical Clearing and Light Sheet Microscopy Imaging of Amphioxus.

Cephalochordates (amphioxi or lancelets) are representatives of the most basally divergent group of the chordate phylum. Studies of amphioxus development and anatomy hence provide a key insight into vertebrate evolution. More widespread use of amphioxus in the evo-devo field would be greatly facilitated by expanding the methodological toolbox available in this model system. For example, evo-devo research on amphioxus requires deep understanding of animal anatomy. Although conventional confocal microscopy can visualize transparent amphioxus embryos and early larvae, the imaging of later developmental stages is problematic because of the size and opaqueness of the animal. Here, we show that light sheet microscopy combined with tissue clearing methods enables exploration of large amphioxus specimens while keeping the surface and the internal structures intact. We took advantage of the phenomenon of autofluorescence of amphioxus larva to highlight anatomical details. In order to investigate molecular markers at the single-cell level, we performed antibody-based immunodetection of melanopsin and acetylated-α-tubulin to label rhabdomeric photoreceptors and the neuronal scaffold. Our approach that combines light sheet microscopy with the clearing protocol, autofluorescence properties of amphioxus, and antibody immunodetection allows visualizing anatomical structures and even individual cells in the 3D space of the entire animal body.

Vessel Network Architecture of Adult Human Islets Promotes Distinct Cell-Cell Interactions In Situ and Is Altered After Transplantation.

Islet-cell hormone release is modulated by signals from endothelial and endocrine cells within the islet. However, models of intraislet vascularization and paracrine cell signaling are mostly based on the rodent pancreas. We assessed the architecture and endocrine cell interaction of the vascular network in unperturbed human islets in situ and their potential to re-establish their endogenous vascular network after transplantation in vivo. We prepared slices of fresh pancreas tissue obtained from nondiabetic patients undergoing partial pancreatectomy. In addition, we transplanted human donor islets into the anterior chamber of the mouse eye. Next, we performed three-dimensional in situ and in vivo imaging of islet cell and vessel architecture at cellular resolution and compared our findings with mouse and porcine islets. Our data reveal a significantly different vascular architecture with decreased vessel diameter, reduced vessel branching, and shortened total vessel network in human compared with mouse islets. Together with the distinct cellular arrangement in human islets, this limits ß to endothelial cell interactions, facilitates connection of α and ß cells, and promotes the formation of independent ß-cell clusters within islets. Furthermore, our results show that the endogenous vascular network of islets is significantly altered after transplantation in a donor age-related mechanism. Thus, our study provides insight into the vascular architecture and cellular arrangement of human islets with apparent consequences for intercellular islet signaling. Moreover, our findings suggest that human islet engraftment after transplantation can be improved by using alternative, less mature islet-cell sources.

Identification of proliferative and mature ß-cells in the islets of Langerhans.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of ß-cells. Pancreatic ß-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature ß-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger ß-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for ß-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional ß-cell heterogeneity and induce ß-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional ß-cell mass in diabetic patients.

Alterations in ß-Cell Calcium Dynamics and Efficacy Outweigh Islet Mass Adaptation in Compensation of Insulin Resistance and Prediabetes Onset.

Emerging insulin resistance is normally compensated by increased insulin production of pancreatic ß-cells, thereby maintaining normoglycemia. However, it is unclear whether this is achieved by adaptation of ß-cell function, mass, or both. Most importantly, it is still unknown which of these adaptive mechanisms fail when type 2 diabetes develops. We performed longitudinal in vivo imaging of ß-cell calcium dynamics and islet mass of transplanted islets of Langerhans throughout diet-induced progression from normal glucose homeostasis, through compensation of insulin resistance, to prediabetes. The results show that compensation of insulin resistance is predominated by alterations of ß-cell function, while islet mass only gradually expands. Hereby, functional adaptation is mediated by increased calcium efficacy, which involves Epac signaling. Prior to prediabetes, ß-cell function displays decreased stimulated calcium dynamics, whereas islet mass continues to increase through prediabetes onset. Thus, our data reveal a predominant role of islet function with distinct contributions of triggering and amplifying pathway in the in vivo processes preceding diabetes onset. These findings support protection and recovery of ß-cell function as primary goals for prevention and treatment of diabetes and provide insight into potential therapeutic targets.

Distinct roles of ß-cell mass and function during type 1 diabetes onset and remission.

Cure of type 1 diabetes (T1D) by immune intervention at disease onset depends on the restoration of insulin secretion by endogenous ß-cells. However, little is known about the potential of ß-cell mass and function to recover after autoimmune attack ablation. Using a longitudinal in vivo imaging approach, we show how functional status and mass of ß-cells adapt in response to the onset and remission of T1D. We demonstrate that infiltration reduces ß-cell mass prior to onset and, together with emerging hyperglycemia, affects ß-cell function. After immune intervention, persisting hyperglycemia prevents functional recovery but promotes ß-cell mass increase in mouse islets. When blood glucose levels return to normoglycemia ß-cell mass expansion stops, and subsequently glucose tolerance recovers in combination with ß-cell function. Similar to mouse islets, human islets exhibit cell exhaustion and recovery in response to transient hyperglycemia. However, the effect of hyperglycemia on human islet mass increase is minor and transient. Our data demonstrate a major role of functional exhaustion and recovery of ß-cells during T1D onset and remission. Therefore, these findings support early intervention therapy for individuals with T1D.

Chimera of IL-2 linked to light chain of anti-IL-2 mAb mimics IL-2/anti-IL-2 mAb complexes both structurally and functionally.

IL-2/anti-IL-2 mAb immunocomplexes were described to have dramatically higher activity than free IL-2 in vivo. We designed protein chimera consisting of IL-2 linked to light chain of anti-IL-2 mAb S4B6 through flexible oligopeptide spacer (Gly(4)Ser)(3). This protein chimera mimics the structure of IL-2/S4B6 mAb immunocomplexes but eliminates general disadvantages of immunocomplexes like possible excess of either IL-2 or anti-IL-2 mAb and their dissociation to antibody and IL-2 at low concentrations. This novel kind of protein chimera is characterized by an intramolecular interaction between IL-2 and binding site of S4B6 mAb similarly as in IL-2/S4B6 mAb immunocomplexes. Our protein chimera has biological activity comparable to IL-2/S4B6 mAb immunocomplexes in vitro, as shown by stimulation of proliferation of purified and activated OT-I CD8(+) T cells. The protein chimera exerts higher stimulatory activity to drive expansion of purified CFSE-labeled OT-I CD8(+) T cells activated by an injection of a low dose of SIINFEKL peptide than IL-2/S4B6 mAb immunocomplexes in vivo.

Antitumor activity of IL-2/anti-IL-2 mAb immunocomplexes exerts synergism with that of N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugate due to its low immunosuppressive activity.

Interleukin (IL)-2 has been approved for treatment of metastatic renal cancer and malignant melanoma. However, its unfavorable pharmacologic properties, severe side effects and the negative role of IL-2 in maintaining T regulatory cells are severe drawbacks. It has been shown that immunocomplexes of IL-2 and certain anti-IL-2 mAbs possess selective and high stimulatory activity in vivo. Here, we show that IL-2/S4B6 mAb immunocomplexes expand not only CD122(high) subsets and newly activated CD8(+) T cells but also natural killer T cells and γδ T cells. Further, we demonstrate that natural killer (NK) cells expanded by IL-2/S4B6 mAb immunocomplexes in vivo have high cytolytic activity, which can be further increased by coadministration of IL-12. We also demonstrate that IL-2/S4B6 mAb immunocomplexes possess noticeable antitumor activity in two syngeneic mouse tumor models, namely BCL1 leukemia and B16F10 melanoma, but only if administered early in tumor progression. To effectively treat established tumors, we administered the tumor-bearing mice first with N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugate, and subsequently with IL-2/S4B6 mAb immunocomplexes alone or with IL-12 to induce an efficient antitumor immune response. Importantly, we show that the conjugate has significantly lower immunosuppressive activity than free doxorubicin when using dosage with comparable antitumor activity, thus eliminating the majority of tumor cells while leaving the immune system mostly unimpaired for stimulation with IL-2/S4B6 mAb immunocomplexes. Indeed, we demonstrate that the conjugate and IL-2/S4B6 mAb immunocomplexes together have synergistic antitumor activity.

In vivo expansion of activated naive CD8+ T cells and NK cells driven by complexes of IL-2 and anti-IL-2 monoclonal antibody as novel approach of cancer immunotherapy.

IL-2 is potent imunostimulatory molecule that plays a key role in T and NK cell activation and expansion. IL-2 is approved by the FDA to treat metastatic renal cancer and melanoma, but its extremely short half-life and serious toxicities are significant limitations of its use. It was reported that in vivo biological activity of IL-2 can be increased by association of IL-2 with anti-IL-2 mAb (S4B6). IL-2/S4B6 mAb immunocomplexes were described to be highly stimulatory for NK and memory CD8(+) T cells and intermediately also for regulatory T cells. IL-2/JES6-1 mAb immunocomplexes are stimulatory solely for regulatory T cells. In this study we show that although both mentioned IL-2 immunocomplexes are less potent than free IL-2 in vitro, they possess extremely high stimulatory activity to expand activated naive CD8(+) T cells in vivo. IL-2 immunocomplexes expand activated naive CD8(+) T cells several hundred-fold times after four doses and more than 1000-fold times after six doses (1.5 microg/dose of IL-2), whereas free IL-2 given at the same dosage shows negligible activity. IL-2/S4B6 mAb immunocomplexes also induce massive expansion of NK cells (40% of DX5(+)NK1.1(+) cells in spleen). Importantly, activated naive CD8(+) T cells expanded by IL-2 immunocomplexes form robust population of functional memory cells. We also demonstrate in two distinct tumor models that IL-2/S4B6 mAb immunocomplexes possess considerable antitumor activity. Finally, by using radioactively labeled IL-2, we provide for first time direct evidence that IL-2 immunocomplexes have much longer half-life in circulation than free IL-2, being approximately 3 h vs <15 min, respectively.

Overcoming immunoescape mechanisms of BCL1 leukemia and induction of CD8+ T-cell-mediated BCL1-specific resistance in mice cured by targeted polymer-bound doxorubicin.

BALB/c mice bearing syngeneic BCL1 leukemia, a mouse model of human chronic lymphocytic leukemia, were treated with polymer-bound doxorubicin conjugate targeted with BCL1-specific monoclonal antibody. Such treatment can cure up to 100% of mice and the cured mice show long-lasting resistance to BCL1 leukemia. We show that both CD4+ and CD8+ T cells are required for establishment of the resistance, but only CD8+ T cells are necessary for its maintenance. BCL1 cells express MHC class I and II and also costimulatory molecules CD80 and CD86, which can aid eliciting of antitumor response. On the other hand, BCL1 cells also use several immunoescape mechanisms, such as expression of PD-L1, PD-L2, and interleukin-10. BCL1 cells thus can be recognized by BCL1-specific T cells, but instead of effective priming, such T cells are anergized or deleted by apoptosis. Moreover, BCL1 leukemia progression is accompanied by robust expansion of CD4+CD25+Foxp3+ regulatory T (Treg) cells. Although it has been shown that depletion of Treg cells in tumor-bearing mice can retard tumor growth, direct evidence that expansion of Treg cells can promote tumor growth was lacking. In this study, we provide first direct evidence that expanded Treg cells can indeed promote tumor progression by using mice with selectively expanded Treg cells before inoculation of BCL1 leukemia. Finally, we have also shown that elimination of some immunoescape mechanism (e.g., deletion of Treg) can significantly improve the therapeutic outcome of chemotherapy.