Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice.

Genetically modified CD8+ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer.

The effectiveness of the oncolytic activity induced by Ad5/F35 adenoviral vector is dependent on the cumulative cellular conditions of survival and autophagy.

To overcome the poor tumor transduction efficiency of adenovirus serotype 5 (Ad5) observed in several types of cancer, the fiber region of Ad5, apart from its tail, was replaced by adenovirus serotype 35 (Ad35). The chimeric Ad5/F35 adenoviral vector did not exhibit any significant enhancement of transduction efficiency. CD46, a receptor for Ad35, was expressed in relatively small amounts in most of the cancer cells examined. Therefore, we investigated the pivotal factor(s) that render cancer cells susceptible to transduction. We discovered that the tumor transduction efficiency of Ad5/F35 was enhanced in the presence of rapamycin, an autophagy inducer, in some cancer cells. Analysis of survival potential and cell proliferation rates revealed that Ad5/F35 exerted a more pronounced oncolytic effect in cancer cells with higher survival potential in the presence of rapamycin.

[In vitro organotypic cultivation of adult newt and rat retinas].

Adult rat and newt retinas were studied during long organotypic 3D cultivation. A high proliferation level was discovered in the region of growth by applying DNA synthesis markers and in vitro mitosis registration in newt retina. Aggregates were formed in the retina spheroid cavity because dedifferentiated cells migrated into this region. Small cell populations in nuclear layers also had dividing and migration capacity. Rosette formation has been shown in newt retina. It is a characteristic of fetal retinal development under pathological conditions. The antiG FAP antibody dye demonstrated an increase in the parent M@uller cell population and generation of a small cell pool with short GFAP-extensions de novo. Recoverin expression studies detected its translocation from photoreceptor extensions to the cell bodies. Moreover, protein was presented in some cells inside the spheroid. It has been shown for the first time that cell proliferation occurred in the developing adult rat retinal spheroid in vitro; BrdU-positive cells and multiple mitoses were revealed in this zone. However, the source of proliferation was not in the peripheral retina, and stable macrophages and glial cells located among neurons of the inner nuclear layer had the ability to divide. The antiGFAP antibody showed an increase in GFAP fibers in the rat retina as well as in the newt retina. Recoverin translocated into photoreceptor perikaryons and the outer plexiform layer in cultivated rat retina. Interestingly, some cells with probably de novo expression of recoverin were discovered in rat and newt retinas.

Larger inhibition of visual pigment kinase in cones than in rods.

In the carp retina, visual pigment kinase, GRK1 (G-protein coupled receptor kinase 1) in rods and GRK7 in cones, is inhibited by a photoreceptor neuronal Ca(2+)-sensor protein, S-modulin (or recoverin) in rods and visinin (formerly named s26) in cones. Here, we compared Ca(2+)-dependent inhibition of GRK1 by S-modulin and that of GRK7 by visinin. First, the concentrations of S-modulin and visinin in the outer segment were estimated: the concentration of visinin (1.2 mM) was 20 times higher than that of S-modulin (53 µM). Based on the determined concentrations of the Ca(2+)-sensor proteins and the known dark Ca(2+) concentrations, we estimated that in situ Ca(2+)-dependent inhibition on GRK in cones would be 2.5 times higher than that in rods at the Ca(2+) concentration in the dark. Because GRK activity is approximately 100 times higher in cones than in rods [Proc. Natl Acad. Sci. USA 102 (2005) 21359], the range of Ca(2+)-dependent inhibition on GRK activity is more than 100 times wider in cones than in rods. The inhibitory effects of S-modulin and visinin on photoreceptor GRKs were indistinguishable, although these Ca(2+)-sensor proteins are expressed in a cell-type specific manner. The inhibition by these Ca(2+)-sensor proteins was slightly higher on GRK7 than GRK1 probably because of a characteristic specific to GRK7.

Establishment of a novel small cell lung carcinoma cell line with specific recoverin expression from a patient with cancer-associated retinopathy.

We analysed the biologic properties of a small cell lung carcinoma cell line (designated KK0206) established from a patient with SCLC who had cancer-associated retinopathy (CAR). Morphological and immunohistochemical studies showed that KK0206 cells have features of the classic type of SCLC. KK0206 cells grew in suspension, forming relatively small clumps of cells with a doubling time of 72 h. On light microscopy, the cells were relatively small with little cytoplasm. On immunohistochemistry using anti-bovine recoverin rabbit antibody, the cells were intensely positive for recoverin. In addition, they were positive for NSE, Ki-67, and TP53. They also expressed human recoverin, a photoreceptor protein, whose presence was confirmed by RT-PCR analysis with cDNA sequencing and Western blot analysis. The point mutation of their TP53 gene (exon 156) was detected as well. The present study demonstrates that human recoverin is expressed in SCLC cells cultured from an anti-recoverin antibody-negative patient with CAR. KK0206 might be important for further research on SCLC related retinopathy.