Growth Stimulation of Tumor-Specific Cytotoxic T Lymphocytes on Concanavalin A-Immobilized Carrier Beads

Human tumor-specific CD4⁺ cytotoxic T lymphocytes (CTL) were generated against duodenum papilloma cell line TGBC18TKB from HLA type-matched peripheral blood mononuclear cells. Concanavalin A (Con A) immobilized on carrier beads stimulated growth of the CTL in a long-term culture without repeated antigen stimulation, while soluble Con A induced death of the CTL. The CTL exhibited the target-specific cytotoxicity in a more potent manner than those before the long-term culture in the presence of the immobilized Con A. Enhanced expression of the adhesion molecule, CD11b, was observed on the CTL. These results suggest that immobilized Con A will be useful for continuous growth stimulation and large scale expansion of CTL without tumor antigen.     

Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Granule-associated perforin and granzymes (gzms) are key effector molecules of cytotoxic T lymphocytes (Tc cells) and natural killer cells and play a critical role in the control of intracellular pathogens and cancer. Based on the notion that many gzms, including A, B, C, K, H, and M exhibit cytotoxic activity in vitro, all gzms are believed to serve a similar function in vivo. However, more recent evidence supports the concept that gzms are not unidimensional but, rather, possess non-cytotoxic potential, including stimulation of pro-inflammatory cytokines and anti-viral activities. The present study shows that isolated mouse gzmB cleaves the actin-severing mouse protein, cytoplasmic gelsolin (c-gelsolin) in vitro. However, when delivered to intact target cells by ex vivo immune Tc cells, gzmB mediates c-gelsolin proteolysis via activation of caspases 3/7. The NH₂-terminal c-gelsolin fragment generated by either gzmB or caspase 3 in vitro constitutively severs actin filaments without destroying the target cells. The observation that gzmB secreted by Tc cells initiates a caspase cascade that disintegrates the actin cytoskeleton in target cells suggests that this intracellular process may contribute to anti-viral host defense.     

Optogenetic Control of Mouse Outer Hair Cells

Normal hearing in mammals depends on sound amplification by outer hair cells (OHCs) presumably by their somatic motility and force production. However, the role of OHC force production in cochlear amplification and frequency tuning are not yet fully understood. Currently, available OHC manipulation techniques for physiological or clinical studies are limited by their invasive nature, lack of precision, and poor temporal-spatial resolution. To overcome these limitations, we explored an optogenetic approach based on channelrhodopsin 2 (ChR-2), a direct light-activated nonselective cation channel originally discovered in Chlamydomonas reinhardtii. Three approaches were compared: 1) adeno-associated virus-mediated in utero transfer of the ChR-2 gene into the developing murine otocyst, 2) expression of ChR-2(H134R) in an auditory cell line (HEI-OC1), and 3) expression of ChR-2 in the OHCs of a mouse line carrying a ChR-2 conditional allele. Whole cell recording showed that blue light (470 nm) elicited the typical nonselective cation current of ChR-2 with reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types. In addition, pulsed light stimulation (10 Hz) elicited a 1:1 repetitive depolarization and ChR-2 currents in mouse OHCs and HEI-OC1 cells, respectively. The time constant of depolarization in OHCs, 1.45 ms, is 10 times faster than HEI-OC1 cells, which allowed light stimulation up to rates of 10/s to elicit corresponding membrane potential changes. Our study demonstrates that ChR-2 can successfully be expressed in mouse OHCs and HEI-OC1 cells and that these present a typical light-sensitive current and depolarization. However, the amount of ChR-2 current induced in our in vivo experiments was insufficient to result in measurable cochlear effects.     

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.     

Tumor-Specific Transplantation Antigen Activity of Freshly Adenovirus-Infected Cells

Newborn hamsters were inoculated with human adenovirus type 12 (Ad12) within 24 hr of birth for tumor induction, and 15 days later, intercurrently immunized with Ad12-infected cells (KB; HeLa; FL; HEK; MoE; HaE). Tumor development was then observed for 75 days thereafter. Tumor formation was prevented at a statistically significant level by immunization with any of the above-mentioned infected cells. The immunization was effective even with abortively infected cells (HaE; MoE) or with cells infected in the presence of 5-fluorodeoxyuridine. The induced immunity was Ad12-specific, since neither cells infected with Ad2, Ad7 or Ad18 nor CV-1 cells infected with SV40 were able to prevent tumor formation. The most plausible explanation to these findings could be that Ad12-specific tumor-specific transplantation antigen is induced on the surface of freshly virus-infected cells and it is responsible for induction of specific cellular immunity. This gives an experimental support to our hypothesis on the mechanism of induction of cellular immunity against virus infections and to the hypothesis proposed by Habel and by Sjögren to explain the immunoresistance against tumor cells induced following viral immunization.     

Spontaneous focal activation of invariant natural killer T (iNKT) cells in mouse liver and kidney

Background  

Invariant natural killer T (iNKT) cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity in vivo has so far been reported.     

Follicular Regulatory T Cells Can Access the Germinal Center Independently of CXCR5

1. Download : Download high-res image (168KB)
2. Download : Download full-size image

     

Flow Cytoenzymology of the Early Differentiation of Mouse Embryonal Carcinoma Cells

Dual parameter flow cytoenzymology was used to detect biochemical differentiation of embryonal carcinoma cells, the undifferentiated, multipotent stem cells of teratocarcinomas. With the use of fluorogenic substrates, two enzyme systems, alkaline phosphatase (EC 3.1.3.1.) and carboxyl esterase (EC 3.1.1.), were studied. Embryonal carcinoma cells passaged in vitro for several years retained high alkaline phosphatase activities similar to those of embryonal carcinoma cells in embryoid bodies grown in vivo. Similar to the embryonal carcinoma cells in vivo, the in vitro embryonal carcinoma cells were capable of giving rise to progeny with greatly decreased levels of alkaline phosphatase. The embryonal carcinoma cell alkaline phosphatase was inhibited by l-p-bromotetramisole, suggesting a relationship between this enzyme and somatic, nonintestinal alkaline phosphatase isoenzymes. Determinations of esterase activities in viable teratocarcinoma cells showed that prior to any evidence of morphologic differentiation, the embryonal carcinoma cells are quite heterogeneous with regard to esterase activities.     

Target Antigen of Vaccinia-Infected Cells Recognized by Virus-Specific Cytotoxic T Lymphocytes

A vaccinia-specific target antigen for recognition of anti-vaccinia cytotoxic T lymphocytes (CTL) was found to be formed on the surface of infected cells through two distinct processes. In the first phase, the expression of the target antigen was dependent on the dose of inoculated virus, without specific protein synthesis. The target antigen seems to be produced by virus-cell fusion. In the second phase, the expression of the target antigen was accompanied by synthesis of an early protein. In spite of the difference in their mode of expression, the first-phase and the second-phase target antigens were cross-reactive in cytotoxicity inhibition assays. Cowpox virus, CPR Cl strain, brought about a lower response than vaccinia virus, IHD-J strain, in both sensitization of CTL and formation of CTL-susceptible cells at both the first and the second phase. The cross-reactive, but inefficient, recognition of anti-vaccinia CTL for cowpox-infected cells suggested a slight difference in the target antigens of the two viruses. Attempts to identify the target antigen were then made by comparing the polypeptide composition of vaccinia virus, cowpox virus, and their recombinants. SDS-PAGE analysis of trypsinactivated viruses revealed 44K (cowpox)/45K (vaccinia) polypeptides which corresponded to the difference in target cell formation. Trypsinization of the viruses also increased the ability of the virus to induce the production of CTL-susceptible target cells.     

Internalization of Embryonal Carcinoma Cells when Aggregated with Normal Mouse Embryos

In the present study, we examined in detail the process of forming chimeric blastocysts between B242g embryonal carcinoma (EC) cells and normal mouse embryos. Electron microscopic observations of the developing aggregates revealed that the embryonic cells spread over the surface of the EC cells, resulting in the internalization of EC cells in the aggregates. When a single blastomere of an 8-cell embryo was aggregated with EC cells, the blastomere spread over and engulfed the EC cells. These results strongly suggest that EC cells are segregated and become situated in the inside position as the development of an aggregate proceeds, and then they are incorporated into the ICM of a blastocyst.     

An Immunocompetent Mouse Model for MLL/AF9 Leukemia Reveals the Potential of Spontaneous Cytotoxic T-Cell Response to an Antigen Expressed in Leukemia Cells

Leukemia differs substantially with respect to stromal milieu from tumors that progress locally as solid masses, and the physiological importance of immunosurveillance in leukemia remains unclear. However, currently available mouse leukemia models have critical limitations in the context of analyzing immunological regulation of leukemia development. In this study, we transferred mouse MLL/AF9 leukemia-initiating cells into immunocompetent recipient mice without any pre-conditioning such as irradiation, and then analyzed the spontaneous T cell response to an immunogenic antigen expressed in leukemia cells. When the minimum numbers of leukemia-initiating cells for engraftment were transferred, leukemia cells were eradicated by the adaptive immune response in most, if not all, wild-type mice, but not in Rag2 ^-/- recipient mice, which lack adaptive immunity. By contrast, mice transplanted with larger numbers of leukemia cells always developed leukemia. In mice with advanced leukemia, antigen-specific CTLs were also expanded, but were unresponsive to antigen stimulation and expressed high levels of PD-1 and LAG-3. These results provide the first clear demonstration that the spontaneous CTL response to a tumor-cell antigen has the potential to eradicate leukemia, whereas antigen-specific CTLs are exhausted in animals with advanced leukemia. This immunocompetent mouse leukemia model provides a useful platform for developing effective immunotherapies against leukemia.     

A General Functional Response of Cytotoxic T Lymphocyte-Mediated Killing of Target Cells

Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and tumor cells, and play a critical role in immune protection. Our knowledge of how the CTL killing efficiency varies with CTL and target cell numbers is limited. Here, we simulate a region of lymphoid tissue using a cellular Potts model to characterize the functional response of CTL killing of target cells, and find that the total killing rate saturates both with the CTL and the target cell densities. The relative saturation in CTL and target cell densities is determined by whether a CTL can kill multiple target cells at the same time, and whether a target cell can be killed by many CTLs together. We find that all the studied regimes can be well described by a double-saturation (DS) function with two different saturation constants. We show that this DS model can be mechanistically derived for the cases where target cells are killed by a single CTL. For the other cases, a biological interpretation of the parameters is still possible. Our results imply that this DS function can be used as a tool to predict the cellular interactions in cytotoxicity data.     

A General Functional Response of Cytotoxic T Lymphocyte-Mediated Killing of Target Cells

Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and tumor cells, and play a critical role in immune protection. Our knowledge of how the CTL killing efficiency varies with CTL and target cell numbers is limited. Here, we simulate a region of lymphoid tissue using a cellular Potts model to characterize the functional response of CTL killing of target cells, and find that the total killing rate saturates both with the CTL and the target cell densities. The relative saturation in CTL and target cell densities is determined by whether a CTL can kill multiple target cells at the same time, and whether a target cell can be killed by many CTLs together. We find that all the studied regimes can be well described by a double-saturation (DS) function with two different saturation constants. We show that this DS model can be mechanistically derived for the cases where target cells are killed by a single CTL. For the other cases, a biological interpretation of the parameters is still possible. Our results imply that this DS function can be used as a tool to predict the cellular interactions in cytotoxicity data.     

Collagen Synthesis in Mouse Embryonal Carcinoma Cells: Effect of Retinoic Acid

Abstract. In five lines of mouse embryonal carcinoma cells, PCC3/A1, PCC4, PCC4/Aza-R1, PCC7-S/Aza-R1, and F9, collagen synthesis was examined by immunofluorescence reaction using specific antibodies directed against collagen. All the embryonal carcinoma cell lines showed type IV collagen, and PCC7-S/Aza-R1 revealed the additional presence of type III collagen. When the F9 and PCC3/A1 EC cells were treated with retinoic acid and dibutyryl-cAMP, they differentiated into morphologically different cellular types. These cellular types showed new types of collagen. Thus, in treated F9 cells, type I, type III, and type V collagen were detected and in treated PCC3/A1 cells, type III and type V collagen were detected.
In two established cellular strains, PYS-2 corresponding to parietal endoderm and 3TDM-1 corresponding to trophoblastoma, collagen was identified by immunological reaction and electrophoretic mobility. The trophoblastoma cell line was characterized by the production of type I, type III, and type IV collagen, whereas endodermal PYS-2 revealed type IV collagen.     

Celecoxib-Induced Cytotoxic Effect Is Potentiated by Inhibition of Autophagy in Human Urothelial Carcinoma Cells

Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, can elicit anti-tumor effects in various malignancies. Here, we sought to clarify the role of autophagy in celecoxib-induced cytotoxicity in human urothelial carcinoma (UC) cells. The results shows celecoxib induced cellular stress response such as endoplasmic reticulum (ER) stress, phosopho-SAPK/JNK, and phosopho-c-Jun as well as autophagosome formation in UC cells. Inhibition of autophagy by 3-methyladenine (3-MA), bafilomycin A1 or ATG7 knockdown potentiated celecoxib-induced apoptosis. Up-regulation of autophagy by rapamycin or GFP-LC3B-transfection alleviated celecoxib-induced cytotoxicity in UC cells. Taken together, the inhibition of autophagy enhances therapeutic efficacy of celecoxib in UC cells, suggesting a novel therapeutic strategy against UC.     

iNKT Cells Suppress the CD8(+) T Cell Response to a Murine Burkitt's-Like B Cell Lymphoma

The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8(+) T cell response. Lymphoma cells transplanted into syngeneic wild type (WT) mice or Jalpha18(-/-) mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8(+) T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18(-/-) mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8(+) T cells. In contrast, lymphoma growth in CD1d1(-/-) mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8(+) T cell response to B cell lymphoma by opposing the effects of type II NKT cells.     

LPS Nephropathy in Mice Is Ameliorated by IL-2 Independently of Regulatory T Cells Activity

Immunosuppressive regulatory T cells (Tregs) have been hypothesized to exert a protective role in animal models of spontaneous (Buffalo/Mna) and/or drug induced (Adriamycin) nephrotic syndrome. In this study, we thought to define whether Tregs can modify the outcome of LPS nephropathy utilizing IL-2 as inducer of tissue and circulating Tregs. LPS (12 mg/Kg) was given as single shot in C57BL/6, p2rx7^−/− and Foxp3^EGFP; free IL-2 (18.000 U) or, in alternative, IL-2 coupled with JES6-1 mAb (IL-2/anti-IL-2) were injected before LPS. Peripheral and tissue Tregs/total CD4+ cell ratio, urinary parameters and renal histology were evaluated for 15 days. IL-2 administration to wild type mice had no effect on peripheral Tregs number, whereas a significant increase was induced by the IL-2/anti-IL-2 immunocomplex after 5 days. Spleen and lymph nodes Tregs were comparably increased. In p2rx7^−/− mice, IL-2/anti-IL-2 treatment resulted in increase of peripheral Tregs but did not modify the spleen and lymph nodes quota. LPS induced comparable and transient proteinuria in both wild type and p2rx7^−/− mice. Proteinuria was inhibited by co-infusion of human IL-2, with reduction at each phase of the disease (24 −48 and 72 hours) whereas IL-2/anti-IL-2 produced weaker effects. In all mice (wild type and p2rx7^−/−) and irrespective of treatment (IL-2, IL-2/anti-IL-2), LPS was associated with progressive signs of renal pathologic involvement resulting in glomerulosclerosis. In conclusion, IL-2 plays a transient protective effect on proteinuria induced by LPS independent of circulating or tissue Tregs but does not modify the outcome of renal degenerative renal lesions.     

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37°C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37°C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.Open in a separate windowClick here to view.^{(75M, flv)}