Analysis of photoreceptor outer segment morphogenesis in zebrafish ift57, ift88 and ift172 intraflagellar transport mutants

Vertebrate photoreceptors are polarized cells that consist of a specialized sensory structure termed the outer segment required for phototransduction and an inner segment that contains the cellular organelles. Proteins synthesized in the inner segment are transported to the outer segment via a connecting cilium by a process called Intraflagellar Transport (IFT). The IFT mechanism refers to the movement of a multisubunit complex along the flagellar axoneme, and mutations in some IFT components cause retinal degeneration. To better understand the role of IFT in early photoreceptor development, we studied zebrafish with mutations in genes encoding three specific subunits of the IFT particle: IFT57, IFT88 and IFT172. These mutants exhibit photoreceptor defects by five days post fertilization (dpf); however, it is not known whether outer segment formation initiates at earlier time points and then degeneration occurs or if outer segments never form at all. To understand this, we performed transmission electron microscopy to study the ultrastructure of photoreceptors at 60, 72 and 96 hours post fertilization (hpf). At 60 hpf, developing outer segments were seen in IFT57 mutant and wild type embryos, however, disorganized membranous structures were observed in IFT88 and IFT172 mutants. At 72 hpf, the number of outer segments in the IFT57 mutants was reduced by 88% when compared to wild type, indicating a defect in initiation of outer segment formation. By 96 hpf we see a reduction in both outer segment length and number in IFT57 mutants. In comparison, the IFT88 and IFT172 mutants do not grow outer segments at any time point. To complement our ultrastructural analysis, we performed immunohistochemistry to understand cell morphology and protein trafficking in these mutants. Zpr1, a marker for cone morphology, showed the presence of normal cones initially that began to degenerate at later time points. Immunohistochemistry with rhodopsin, a phototransduction protein that localizes to the outer segment, revealed that rhodopsin was mislocalized in all the three mutants by 96 hpf. Connecting cilia labeled with acetylated tubulin were highly reduced in IFT57 mutants whereas none were observed in IFT88 and IFT172 mutants. Together these data indicate that IFT57 is required for maintenance and growth of outer segments whereas IFT88 and IFT172 are required for initiating outer segment formation

Analysis of photoreceptor outer segment morphogenesis in zebrafish ift57, ift88 and ift172 intraflagellar transport mutants

Vertebrate photoreceptors are polarized cells that consist of a specialized sensory structure termed the outer segment required for phototransduction and an inner segment that contains the cellular organelles. Proteins synthesized in the inner segment are transported to the outer segment via a connecting cilium by a process called Intraflagellar Transport (IFT). The IFT mechanism refers to the movement of a multisubunit complex along the flagellar axoneme, and mutations in some IFT components cause retinal degeneration. To better understand the role of IFT in early photoreceptor development, we studied zebrafish with mutations in genes encoding three specific subunits of the IFT particle: IFT57, IFT88 and IFT172. These mutants exhibit photoreceptor defects by five days post fertilization (dpf); however, it is not known whether outer segment formation initiates at earlier time points and then degeneration occurs or if outer segments never form at all. To understand this, we performed transmission electron microscopy to study the ultrastructure of photoreceptors at 60, 72 and 96 hours post fertilization (hpf). At 60 hpf, developing outer segments were seen in IFT57 mutant and wild type embryos, however, disorganized membranous structures were observed in IFT88 and IFT172 mutants. At 72 hpf, the number of outer segments in the IFT57 mutants was reduced by 88% when compared to wild type, indicating a defect in initiation of outer segment formation. By 96 hpf we see a reduction in both outer segment length and number in IFT57 mutants. In comparison, the IFT88 and IFT172 mutants do not grow outer segments at any time point. To complement our ultrastructural analysis, we performed immunohistochemistry to understand cell morphology and protein trafficking in these mutants. Zpr1, a marker for cone morphology, showed the presence of normal cones initially that began to degenerate at later time points. Immunohistochemistry with rhodopsin, a phototransduction protein that localizes to the outer segment, revealed that rhodopsin was mislocalized in all the three mutants by 96 hpf. Connecting cilia labeled with acetylated tubulin were highly reduced in IFT57 mutants whereas none were observed in IFT88 and IFT172 mutants. Together these data indicate that IFT57 is required for maintenance and growth of outer segments whereas IFT88 and IFT172 are required for initiating outer segment formation

Early defects in photoreceptor outer segment morphogenesis in zebrafish ift57, ift88 and ift172 Intraflagellar Transport mutants

AbstractIntraflagellar Transport (IFT) refers to a highly conserved process occurring in eukaryotic ciliated structures. In vertebrate photoreceptors, IFT mediates protein trafficking to the outer segments. The IFT particle is a multi-subunit complex and mutations in many individual components causes photoreceptor defects. In zebrafish, mutations in the ift57, ift88, and ift172 genes result in retinal degeneration by 5 days post fertilization (dpf). Although the effects of these mutations on photoreceptor survival have been described, early developmental morphogenesis remains poorly understood. We used transmission electron microscopy and immunohistochemistry to examine these mutants at 60, 72, and 96h post fertilization (hpf) and describe early photoreceptor morphogenesis defects

Engineered CAR T cell therapy for solid tumors

The adoptive transfer of T cells redirected to tumor-associated antigens via transgenic expression of chimeric antigen receptors (CARs) has produced impressive clinical responses in patients with hematologic malignances. However the successful extension of this therapy to solid tumors has proven challenging due to i) the paucity of target antigens that are tumor selective, leading to a heightened risk of “on-target, off-tumor” toxicities and, ii) the suppressive tumor microenvironment, which subverts T cell effector function. Therefore, to overcome these limitations we have programmed T cells with a combination of receptors that recognize a gene expression pattern that is unique to the tumor site and whose endodomains deliver intracellular signals 1, 2 and 3 (antigen, co-stimulation and cytokine) required for optimal T cell activation and protection from suppressive factors present at the tumor site. The current presentation will not only highlight our T cell engineering improvements but also our process optimization, including the incorporation of the G-Rex device, to facilitate the clinical and commercial development of potentially curative therapie

CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation

Abstract Background The adoptive transfer of T cells redirected to tumor via chimeric antigen receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to breast cancer, we generated CAR T cells directed against mucin1 (MUC1), an aberrantly glycosylated neoantigen that is overexpressed by malignant cells and whose expression has been correlated with poor prognosis. Furthermore, to protect our tumor-targeted cells from the elevated levels of immune-inhibitory cytokines present in the tumor milieu, we co-expressed an inverted cytokine receptor linking the IL4 receptor exodomain with the IL7 receptor endodomain (4/7ICR) in order to transform the suppressive IL4 signal into one that would enhance the anti-tumor effects of our CAR T cells at the tumor site. Methods First (1G - CD3ζ) and second generation (2G - 41BB.CD3ζ) MUC1-specific CARs were constructed using the HMFG2 scFv. Following retroviral transduction transgenic expression of the CAR±ICR was assessed by flow cytometry. In vitro CAR/ICR T cell function was measured by assessing cell proliferation and short- and long-term cytotoxic activity using MUC1+ MDA MB 468 cells as targets. In vivo anti-tumor activity was assessed using IL4-producing MDA MB 468 tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results In the IL4-rich tumor milieu, 1G CAR.MUC1 T cells failed to expand or kill MUC1+ tumors and while co-expression of the 4/7ICR promoted T cell expansion, in the absence of co-stimulatory signals the outgrowing cells exhibited an exhausted phenotype characterized by PD-1 and TIM3 upregulation and failed to control tumor growth. However, by co-expressing 2G CAR.MUC1 (signal 1 - activation + signal 2 - co-stimulation) and 4/7ICR (signal 3 - cytokine), transgenic T cells selectively expanded at the tumor site and produced potent and durable tumor control in vitro and in vivo. Conclusions Our findings demonstrate the feasibility of targeting breast cancer using transgenic T cells equipped to thrive in the suppressive tumor milieu and highlight the importance of providing transgenic T cells with signals that recapitulate physiologic TCR signaling – [activation (signal 1), co-stimulation (signal 2) and cytokine support (signal 3)] - to promote in vivo persistence and memory formation

Additional file 1: of CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation

Figure S1. Generation of 2nd generation CAR.MUC1 T cells. (A) Schematic of 2nd generation CAR.MUC1 (2G) retroviral construct. (B) Co-expression of 4/7ICR and 2G CAR as detected by mOrange and anti-IgG, respectively. Summary data (right panel) shows percentage of double-positive cells (mean ± SEM, n = 4). (PPTX 298 kb