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… Antibody Response of Mice to Tumour Inoculation

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... Therefore, we gain a deeper understanding of the Mhc as we explore it in molecular and cellular terms. MHC molecules are cell surface receptors that bind antigen fragments and display them to various cells of the immune system, T cells that bear αβ receptors (10, 11,12), NK cells (13)(14)(15), T cells that express γ δ receptors (16), and NKT cells (17)(18)(19)(20). Molecules that are structurally similar to MHC-I molecules but are encoded beyond the strict genetic bounds of the Mhc, and in some cases lacking the full complement of MHC-I domains, are now known to be expressed on particular subsets of somatic cells, some tumors, and cells of the placenta. ...
... For the genes of the human Mhc this convention is often overlooked, whereas for those of the mouse and other species it is frequently followed. The mouse Mhc is referred to as H2 because it was the second genetic locus involved in control of expression of erythrocyte antigens to be identified by Gorer (18,19). Now, the Mhc is known to consist of many loci, and the extended genetic region is referred to as the complex; thus, the general term used for all species is the Mhc or MHC. ...
... They were cultivated in RPMI plus 10% AB human serum (Gibco, Life Technologies, Monza, Italy), and regularly monitored for its specificity. Both mouse splenocytes and EL-4 cells, i.e., murine thymic lymphoma CD4 + T cells originally obtained from C57 Bl/6 mice upon treatment with 9,10-dimethyl-1,2-benzanthracene [28], were cultivated in RPMI medium supplemented with 10% FCS. ...
Article
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We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.
... Recombinant antibodies, termed B2-IgM and B2-IgG1, were purified from the culture supernatants of VBCM or VBCG1 cells on a NP-bovine serum albumin (BSA) column. A mouse T cell lymphoma , EL4 (Gorer, 1950), was used as a source of cells which do not produce antibodies. Unlabeled, FITC, PE, or alkaline phosphataseconjugated goat polyclonal antibodies to mouse Igλ, IgM, IgG, and IgG1 were purchased from SouthernBiotech. ...
Article
We developed a method to detect and isolate plasma cells that produce antigen-specific antibodies. An affinity matrix of hapten was constructed on a cell surface, and subsequent incubation allowed cells to secrete antibodies. Anti-hapten antibodies preferentially bound to the affinity matrix on the cells from which they were secreted. We showed that the combination of surface biotinylation and streptavidin which was conjugated with a high valence of hapten was suitable for sensitive detection of antibody binding. Using this protocol, anti-hapten plasma cells from immunized mouse spleen were detected and enriched by flow cytometry. This method allows for isolation of intact plasma cells according to the antibody specificity and may be useful for highly efficient and precise analysis of an antibody repertoire. Copyright © 2015. Published by Elsevier B.V.
... The many differences may reflect that the cell line was heterogeneous when first developed in 1945, and that this heterogeneity has persisted and likely expanded over time. In the original report characterizing EL4 cells, the author commented that the phenotype of the cells, as seen by gross morbid anatomy, shifted from "lymphatic leukemia" to "lymphosarcoma" when they were propagated by intraperitoneal injection (1). Thus, different cell types in the original line could have been subject to selection under different growth conditions. ...
Article
Background: Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. Materials and methods: Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. Results: Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). Conclusion: The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells.
... The focus of this investigation is the mouse lymphoblastic lymphoma cell line (EL4) lymphoma-derived tumor model in C57BL/6 mice. The murine EL4 lymphoma cell line was first established in 1945, following treatment of C57BL/6 mice with 9:10-dimethyl-1:2-benzanthracene [1] and has since become a widely utilized model for investigating lymphoma tumors and tumor apoptosis in vivo. Having originated in C57BL/6 mice, EL4 cellular suspensions can be injected into these animals without inducing immunological tissue rejection [2][3][4]. ...
Article
The murine EL4 tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research little is known regarding the molecular details of the in vivo tumor cell death. Here we report the first in-depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label-free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the down regulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected down regulation of caspase 3 and an up regulation of caspase 6 in addition to a global up regulation of lysosomal proteins in the bulk of the tumor. This article is protected by copyright. All rights reserved.
... The sex of the mouse from which the J558L myeloma cell line was derived has not been reported (Oi et al., 1983). EL4 are a mouse thymic lymphoma cell-line (Gorer, 1950). The sex of the mouse from which the EL4 cell line was derived has not been reported. ...
Article
Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRβ signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.
... Cells were cultured in RPMI 1640 with 10% fetal bovine serum (GIBCO). EL4 cell line was established from a lymphoma induced in a C57BL/6J mouse by 9,10dimethyl1,2benzan thracene (18). Cells were cultured at an optimal concentration (1-5 × 10 5 cells/ml) in RPMI1640 medium supplemented with 10% v/v fetal bovine serum, 2 mmol/l glutamine, and 100 mg/ ml streptomycin (all from Life Technologies), at 37°C in 5% CO2 atmosphere, as previously described (19). ...
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Clinical data and experimental studies have suggested a relationship between psychosocial factors and cancer prognosis. Both, stress effects on the immune system and on tumor biology were analyzed independently. However, there are few studies regarding the stress influence on the interplay between the immune system and tumor biology. Moreover, antidepressants have been used in patients with cancer to alleviate mood disorders. Nevertheless, there is contradictory evidence about their action on cancer prognosis. In this context, we investigated the effect of chronic stress on tumor progression taking into account both its influence on the immune system and on tumor biology. Furthermore, we analyzed the action of selective serotonin reuptake inhibitors, fluoxetine and sertraline, in these effects. For this purpose, C57BL/6J mice submitted or not to a chronic stress model and treated or not with fluoxetine or sertraline were subcutaneously inoculated with EL4 cells to develop solid tumors. Our results indicated that chronic stress leads to an increase in both tumor growth and tumor cell dissemination. The analysis of cell cycle regulatory proteins showed that stress induced an increase in the mRNA levels of cyclins A2, D1, and D3 and a decrease in mRNA levels of cell cycle inhibitors p15, p16, p21, p27, stimulating cell cycle progression. Moreover, an augment of mRNA levels of metalloproteases (MMP-2 and MMP-9), a decrease of inhibitors of metalloproteases mRNA levels (TIMP 1, 2, and 3), and an increase in migration ability were found in tumors from stressed animals. In addition, a significant decrease of antitumor immune response in animals under stress was found. Adoptive lymphoid cell transfer experiments indicated that the reduced immune response in stressed animals influenced both the tumor growth and the metastatic capacity of tumor cells. Finally, we found an important beneficious effect of fluoxetine or sertraline treatment on cancer progression. Our results emphasize the crucial role of the immune system in tumor progression under stress situations. Although a direct effect of stress and drug treatment on tumor biology could not be ruled out, the beneficial effect of fluoxetine and sertraline appears to be mainly due to a restoration of antitumor immune response.
... To this end, an hgp100 minigene sequence was constructed encoding the minimal epitope along with 16 amino acids of endogenous flanking sequence and inserted into the minigene-FRET lentiviral cassette in similar fashion to the Ova minigenes described above. After virus production, both hgp100 minigene and Ova minigene constructs were transduced into the EL4 cell line (Gorer, 1950), a mouse T-cell lymphoma induced in the C57BL/6 strain. ...
Thesis
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Though it is understood that T-cells are a critical component of the immune system’s ability to destroy foreign invaders and attack cancerous cells, very little is known regarding the specific epitopes that are recognized by T-cells to carry out these functions. Generally, the epitopes mediating this immunity are short, contiguous peptides derived from antigenic proteins presented on major histocompatibility complex (MHC) molecules for inspection by T-cells. The ability to rapidly and deeply search peptide space to determine specific peptide epitopes that are naturally processed, presented, and capable of eliciting functional T-cell responses is a critical unmet need in the study of adaptive immunity. Here, I describe a novel method for deep T-cell epitope profiling that enables simultaneous in vitro interrogation of target cell populations encoding high-diversity minigene libraries with T-cell populations-of-interest. Targets eliciting T-cell reactivity are selectively isolated by fluorescence-activated cell sorting (FACS) and identified by deep amplicon sequencing. The approach was extensively validated using known murine T-cell receptor (TCR)/peptide-MHC pairs and it was shown that this method can unambiguously identify canonical minigenes from libraries of vastly more candidate antigens in parallel than would be feasibly tractable using conventional methods. The capability of this strategy was extended by applying a synthetic biology approach. Using pairs of immortalized natural killer (NK)-like effector cell lines and naturally tolerated target cell lines, I showed that fully reconstituting the TCR/CD3 complex in effectors and expressing relevant MHC-/minigene-coding sequences in targets is sufficient to re-direct the cytotoxicity of NK-like cell lines towards antigenic targets of recombinant TCR. These results provide indication that it should be possible to use an entirely synthetic framework for functionally screening recombinant TCR-of-interest against minigene libraries without the requirement to first isolate and expand primary T-cell clones or donor-derived antigen-presenting cells. The high-throughput T-cell antigen profiling methods described and validated in this thesis could allow investigators to generate TCR epitope data broader in scope than previously possible to better understand basic T-cell biology, develop better predictive models of T-cell reactivity, and rationally design T-cell-based immunotherapeutics for the treatment of cancer, infectious disease, and autoimmunity.
... Tumors. EL4 and EL4-OVA (also known as EG7) cells were obtained from ATCC (47,48). LLC cells were obtained from Douglas Fearon (Cancer Research UK Cambridge Institute, Cambridge, United Kingdom) along with the LLC-OVA derivative (26). ...
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Multiple modes of immunosuppression restrain immune function within tumors. We previously reported that phosphoinositide 3-kinase δ (PI3Kδ) inactivation in mice confers resistance to a range of tumor models by disrupting immunosuppression mediated by regulatory T cells (Tregs). The PI3Kδ inhibitor idelalisib has proven highly effective in the clinical treatment of chronic lymphocytic leukemia and the potential to extend the use of PI3Kδ inhibitors to nonhematological cancers is being evaluated. In this work, we demonstrate that the antitumor effect of PI3Kδ inactivation is primarily mediated through the disruption of Treg function, and correlates with tumor dependence on Treg immunosuppression. Compared with Treg-specific PI3Kδ deletion, systemic PI3Kδ inactivation is less effective at conferring resistance to tumors. We show that PI3Kδ deficiency impairs the maturation and reduces the capacity of CD8+ cytotoxic T lymphocytes (CTLs) to kill tumor cells in vitro, and to respond to tumor antigen-specific immunization in vivo. PI3Kδ inactivation antagonized the antitumor effects of tumor vaccines and checkpoint blockade therapies intended to boost the CD8+ T cell response. These findings provide insights into mechanisms by which PI3Kδ inhibition promotes antitumor immunity and demonstrate that the mechanism is distinct from that mediated by immune checkpoint blockade.
... To study whether IVIg can also interfere directly with the cytotoxicity of activated CD8 + T cells, OT-I cells were first activated by SIINFEKL peptide-pulsed wild-type splenocytes in the absence of IVIg during 3 days. In parallel, EL4 cells (derived from a lymphoma induced in C57BL/6 mice) 26 were stained with a viability dye, pulsed with the SIINFEKL peptide and used as target cells in a cytotoxicity assay that was performed in the presence or absence of IVIg. The pulsed EL4 cells were added to the OVA-activated OT-I cells. ...
Article
Intravenous immunoglobulin (IVIg) is successfully used in the treatment of autoimmune diseases involving self-reactive CD8+ T cells. However, its direct influence on the cytotoxic response remains unknown. Using an antigen cross-presentation assay and a mouse model of ovalbumin (OVA) immunization, we showed that IVIg decreases the in vitro activation, proliferation and cytokine secretion of OVA-specific CD8+ T cells (OT-I), as well as the in vivo generation of OVA-specific CD8+ T cells. In addition, IVIg significantly decreases the proportion of perforin- and CD107a-expressing CD8+ T cells, and inhibits the cytotoxic activity of OVA-activated OT-I cells. The interference of IVIg with the CD8+ T cell response is associated with TCR blockade, therefore reducing the interaction between effector and target cells. A similar blockade is observed on human CD8+ T cells, suggesting that the observations reported here could apply to the IVIg-mediated improvement of CD8+ T cell-mediated autoimmune conditions in human patients. This article is protected by copyright. All rights reserved.
... EL4 cells were established in tissue culture from a murine 9:10-dimethyl-benzanthracene induced thymoma from C57BL/6 mice (Gorer, 1950). RMA-S cells originate from a Rauscher virus-induced C57BL/6 T cell lymphoma RBL-5 which was mutagenised with ethyl methane sulfonate (Ljunggren and Karre, 1985). ...
Thesis
For a vertebrate to survive and reproduce, it must be able to fight infection efficiently throughout its life-span. The vertebrate immune system performs this function by recognising, responding to, killing and remembering pathogens. A crucial component of immunity is the dendritic cell, which responds to inflammatory signals such as cytokines which are produced because of infection. The invading pathogen is captured and partially degraded by dendritic cells via a mechanism termed antigen processing. Dendritic cells are specialised to prime specific T cells against small regions (called determinants) derived from the pathogen by antigen processing. The activated T cells then control the immune response which fights the infection. This thesis is a study of how antigen processing by dendritic cells can be affected by cytokines. The determinants processed and presented from a model antigen are shown to vary considerably depending upon which cytokines the dendritic cells are exposed to. In particular, determinants that are normally not presented (cryptic determinants) can become very immunogenic: the cytokine exposed dendritic cells activate T cells against these cryptic deteminants both in vitro and in vivo. How one particular cytokine, interleukin-6, can cause these effects is further investigated. Although the exact mechanism is not elucidated, interleukin-6 is shown to differentiate dendritic cells in novel ways, including acidifying certain intracellular compartments which are involved in antigen processing. Because pH influences many aspects of antigen processing, the display of cryptic determinants by interleukin-6 treated dendritic cells can very probably be accounted for by this characteristic.
... To investigate whether the subcellular distribution of CD93 is dependent on the expression of the BCR-ABL1 oncogene, we assessed the localization of CD93-GFP in BCR-ABL1-negative CD93-expressing EL-4 lymphoma (Gorer, 1950) and STR-4 endothelial BM cells (Aizawa et al., 1991) after transfection with pAcGFP1-N1-mCd93. In contrast to BCR-ABL1-expressing K562 CML cells, the CD93-GFP fusion protein was detected at the cell membrane, but not in the nucleus of EL-4 and STR-4 cells ( Figure 3G). ...
Article
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Self-renewal is a key characteristic of leukemia stem cells (LSCs) responsible for the development and maintenance of leukemia. In this study, we identify CD93 as an important regulator of self-renewal and proliferation of murine and human LSCs, but not hematopoietic stem cells (HSCs). The intracellular domain of CD93 promotes gene transcription via the transcriptional regulator SCY1-like pseudokinase 1 independently of ligation of the extracellular domain. In a drug library screen, we identify the anti-emetic agent metoclopramide as an efficient blocker of CD93 signaling. Metoclopramide treatment reduces murine and human LSCs in vitro and prolongs survival of chronic myeloid leukemia (CML) mice through downregulation of pathways related to stemness and proliferation in LSCs. Overall, these results identify CD93 signaling as an LSC-specific regulator of self-renewal and proliferation and a targetable pathway to eliminate LSCs in CML.
... The murine lymphoma EL4 (17) was grown in RPMI 1640 supplemented with 5% FCS, 2 mM L-glutamine, and 100 U/ml streptomycin/penicillin . The acute T cell leukemia Jurkat (18) was grown in RPMI-5. ...
Article
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Antigenic T cell stimulation requires interaction between the TCR of the T cell and cognate peptide-MHC molecules presented by the APC. Although studies with TCR-specific Abs and soluble peptide-MHC ligands have shown that the TCR needs to be crosslinked by two or more ligands to induce T cell stimulation, it is not understood how several MHC molecules loaded with the cognate antigenic peptide can produce crosslinking under physiological conditions. We show at the molecular level that large clusters of cognate peptide-MHC are formed at the surface of murine professional and nonprofessional APCs upon virus infection and that these clusters impinge on the stimulatory capacity of the APC. These clusters are formed by tight apposition of cognate peptide-MHC complexes in a configuration that is compatible with simultaneous engagement of two or more TCRs. This suggests that physiological expression of Ag allows formation of multivalent ligands for the TCR that permit TCR crosslinking and T cell activation.
Article
The main goal of this study was to assess the potential of the mouse thymoma EL-4 cell line in screening for chemical induced immunotoxicity. Therefore, EL-4 cells were exposed to two well-known immunotoxicants, organotin compound tributyltin oxide (TBTO, 0.5 and 1 μM for 3 or 6 h) and the mycotoxin deoxynivalenol (DON, 0.25, 0.5 and 1 μM for 3, 6 or 11 h). Previous studies in human Jurkat T cells and mouse thymus in vivo showed that the primary mode of action of TBTO is induction of endoplasmic reticulum (ER) stress, T cell activation and apoptosis. DON induces ribotoxic stress and, similarly to TBTO, induces ER stress, T cell activation and apoptosis. In the present study, the effects of TBTO and DON on EL-4 mRNA expression were assessed by whole genome microarray analysis. The microarray data were then compared to those obtained with mouse thymuses in vivo, mouse thymocytes in vitro, and CTLL-2 cells and human Jurkat cells in vitro exposed to TBTO or DON. Analysis at the level of gene sets revealed that part of the previously detected modes of action of TBTO and DON were not observed in the EL-4 cell line. In EL-4 cells, TBTO induced genes involved in calcium signalling and ER stress but did not induce genes involved in T cell activation and apoptosis. DON induced RNA related processes and ribosome biogenesis. Furthermore, DON downregulated ER stress, T cell activation and apoptosis which is opposite to the mechanism of DON observed in the mouse thymus in vivo and in Jurkat T cells in vitro. Apparently, EL-4 cells lack factors that are necessary to link ribotoxic stress to ER stress. In addition, of the lack of T cell activation response of EL-4 cells to TBTO is likely due to the fact that these cells are in a constitutively activated state already. Based on the results obtained for TBTO and DON, it can be concluded that the EL-4 cell line has limited value for immunotoxicogenomics based screening.
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Previously, we showed that an 88-kD protein (p88) associates rapidly and quantitatively with newly synthesized murine major histocompatibility complex class I molecules within the endoplasmic reticulum (ER). This interaction is transient and dissociation of p88 appears to be rate limiting for transport of class I molecules from the ER to the Golgi apparatus. In this report, we examine the relationship between p88 interaction and assembly of the ternary complex of class I heavy chain beta 2-microglobulin (beta 2m), and peptide ligand. In both murine and human beta 2m-deficient cells, in which little or no transport of class I heavy chains is observed, p88 remained associated with intracellular heavy chains throughout their lifetime. In murine RMA-S cells, which are apparently defective in accumulating peptide ligands for class I within the ER, prolonged association of p88 with "empty" heavy chain-beta 2m heterodimers was also observed. However, p88 dissociated slowly in parallel with the slow rate of ER to Golgi transport of empty class I molecules in these cells. The close correlation between p88 association and impaired class I transport suggests that p88 functions to retain incompletely assembled class I molecules in the ER. We propose that conformational changes in class I heavy chains induced by the binding of both beta 2m and peptide are required for efficient p88 dissociation and subsequent class I transport.
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In this report we describe a variant of the C57BL/6 T lymphoma EL4 (EL4/Mar) which, in contrast to the parental cell line, expresses neither H-2Kb nor beta2-microglobulin (beta2m) but which does express H-2Db detectable by serology and by alloreactive cytotoxic T lymphocytes (CTL). This observation raises the possibility that H-2Db and perhaps other major histocompatibility complex class I molecules are normally not associated with beta2m on the cell surface. In addition, this report is the first to indicated that alloreactive CTL can interact with a beta2m-free class I antigen.
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The present study reports the establishment of an NK (natural killer) cell line, designated as IL2-CE1. This cell line was maintained in active growth in culture with the supplementation of interleukin 2 (IL2). The cells gave cytotoxicity against a variety of murine tumors. There was no detectable cytotoxicity against three human tumors tested. The reactivity obtained with the murine tumors was generally correlated with the sensitivity of these tumor cells to NK cytotoxicity. With few exceptions, the reactivity was much higher for NK-sensitive targets like YAC and RL♂ 1 cells. There was very little cytotoxicity against NK-resistant targets like P815 and HFL/d. The reactivity of the effectors against YAC was susceptible to trypsin treatment and this effect was reversible. In determining the surface markers of IL2-CE1 cells, it was shown that over 90% of the cells had Thy-1.2 antigen despite their resistance to the lytic effect of anti-Thy-1.2 antibody. There were no detectable surface Ig or Fc receptors. Therefore, the IL2-CE1 cells were consistent with being NK cells. Although these cells gave high levels of cytotoxicity against murine tumors, in the tumor neutralization tests the IL2-CE1 cells failed to give any protection against an immunosensitive leukemic line, FBL-3. After the IL2-CE1 cells were cloned, it was found that different NK clones showed variations in their fine specificity. Our finding supports the presence of different populations or subsets of NK cells. Establishment of these NK clones in long-term culture should be helpful for the detailed characterization of the NK cells and aid in the determination of their significance in the immune surveillance against neoplasia.
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Lung adenocarcinoma (AC) induced by carcinogen inhalation never came close to the human counterpart. This has changed with the creation of genetically engineered models. Different types of mouse AC were studied, some of them with a KRAS codon12 mutation background, others with HRAS mutants inserted; in most additional hits were introduced (ATG5, PTEN, PI3K, p53). Development of AC was followed for 28 weeks. Human lung AC subtypes and precursor lesions served for comparison. AC in mice starts with papillary growth at the bronchoalveolar junction (baJ). Tumor spreads into the alveolar periphery. Due to different cell to alveolar size alveoli are filled almost completely, simulating solid growth. At a certain size hypoxic necrosis is seen in centers, followed by neoangiogenesis and desmoplastic stroma formation. Finally invasion of tumor cells into stroma occur. Metastasis is rare, due to high tumor load, causing early death. Vascular invasion is seen, if second carcinogenic hits are applied. Dissimilarities with human precursor and AC types are: AC in mice are all non-mucinous, they are predominant papillary or solid, often with a high degree of signet ring cell formation; in addition there seems to be a requirement of large tumor size before invasion and metastasis occur. To understand AC development knowledge of the anatomy and histology of mouse and human lung is necessary, but these models open a new way of investigating lung AC. Citation Format: Helmut H. Popper, Beatrice Grabner, Emilio Casanova, Robert Eferl, Rao Shuan, Josef Penninger. Comparison of lung adenocarcinoma development in genetically engineered mouse and in humans - similarities and differences. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1209. doi:10.1158/1538-7445.AM2014-1209
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The fluorescent antibody technique has been applied for the demonstration of mouse isoantigens at the cellular level. Specific reactions were obtained by the indirect or "sandwich" technique with a variety of living normal and neoplastic cells. Isoantigens of the H-2 system and of other systems could be demonstrated as well and appeared to be localized at the cell membrane. As far as the H-2 system was concerned, the membrane localization could be confirmed on histological sections. Different types of non-specific staining reactions have been identified and described. Pinocytosis and cell injury led to such reactions that were morphologically distinguishable from the specific "ring" reaction and as far as pinocytosis is concerned, could be easily avoided by reducing the incubation time. In addition, a non-specific staining reaction morphologically indistinguishable from the specific "ring" reaction could be seen in a small proportion of bone marrow and lymph node cells but in no other cell type studied. The possible nature of this reaction is discussed.
Article
The relation between serum antibody and resistance to tumor homografts in the mouse has been investigated. Production of serum antibody in response to homografts of a transplantable sarcoma (Sarcoma 1) was demonstrated, by cytotoxic action on the cells of the tumor, and also by a hemagglutinin test. The simpler and more repeatable hemagglutinin test was further investigated. Peak hemagglutinin titres were reached after the immunizing homografts underwent breakdown. Following transfer of lymph node cells from immunized mice into hosts of the same strain, hemagglutinin could be detected in the host serum. The course of its production showed that this secondary antibody was not elicited by transferred antigen, nor could it be due to transfer of preformed antibody. The cells developed the capacity to transfer hemagglutinin production later than the power to transfer heightened graft resistance. Spleen cells also transferred hemagglutinin production, at a later stage after immunization and to a lesser extent than cells from the regional lymph nodes. Implantation of the sarcoma in mice pretreated with certain preparations of lyophilized or frozen tissue stimulated hemagglutinin production, although the tumor grew progressively. The regional lymph nodes participated in the response: they could transfer hemagglutinin production into secondary hosts, but not graft resistance, and indeed appeared to diminish resistance. Lymph node cells from immunized donors conferred protection against the tumor on pretreated mice. Lymph nodes from normal donors also appeared in some experiments to confer protection although the effect was obscured by the rapidity with which the growing tumor became immunologically invulnerable. The fate of lymph node cells stained with acriflavine was followed after transfer. No effect of the staining on the power of the cells to confer immunity could be detected. Cells transferred to the peritoneal cavity passed into various host tissues, but were not found in test homografts. The conclusion is drawn that the hemagglutinating antibody is distinct from the antibody effective in combating homografts. The similarity in this respect between the homograft reaction and sensitization is emphasized in discussion.
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When a tumor evolves, there is constant crosstalk between the transformed cells and cells of the immune system. Transplantation of well-established tumor cell lines into genetically engineered mice is a valuable tool to study the contribution of a gene of interest to tumor surveillance. These methods bear several advantages: first, such cell lines are well characterized; second, much data for reference exist; and third, the impact of the immune system can be separated from tumor cell intrinsic effects. Here, we provide protocols for tumor cell transplantations to address the role of a specific gene product in tumor surveillance. We furthermore describe several approaches to define the impact of natural killer cells and T cells, such as cell depletion and adoptive transfer experiments or use of different genetically modified mice.
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We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.
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The preclinical development of anticancer drugs including immunotherapeutics and targeted agents relies on the ability to detect minimal residual tumor burden as a measure of therapeutic efficacy. Real-time quantitative (qPCR) represents an exquisitely sensitive method to perform such an assessment. However, qPCR-based applications are limited by the availability of a genetic defect associated with each tumor model under investigation. Here, we describe an off-the-shelf qPCR-based approach to detect a broad array of commonly used preclinical murine tumor models. In particular, we report that the mRNA coding for the envelope glycoprotein 70 (gp70) encoded by the endogenous murine leukemia virus (MuLV) is universally expressed in 22 murine cancer cell lines of disparate histological origin but is silent in 20 out of 22 normal mouse tissues. Further, we detected the presence of as few as 100 tumor cells in whole lung extracts using qPCR specific for gp70, supporting the notion that this detection approach has a higher sensitivity as compared with traditional tissue histology methods. Although gp70 is expressed in a wide variety of tumor cell lines, it was absent in inflamed tissues, non-transformed cell lines, or pre-cancerous lesions. Having a high-sensitivity biomarker for the detection of a wide range of murine tumor cells that does not require additional genetic manipulations or the knowledge of specific genetic alterations present in a given neoplasm represents a unique experimental tool for investigating metastasis, assessing antitumor therapeutic interventions, and further determining tumor recurrence or minimal residual disease.
Article
Homografts of all kinds excite the formation of humoral antibodies, but the part played by antibodies in the rejection of skin homografts is still uncertain. Antisera were prepared in mice by the repeated intraperitoneal injection of dissociated lymphoid cells, or by multiple intra-cutaneous injections, on a single occasion, of lymphoid cells incorporated into a myco­bacterial adjuvant. We were unable to discover any circumstances under which the passive transfer of high or low doses of these antisera could shorten the life of freshly transplanted skin homografts in mice (§3). Antisera were equally without effect on skin homografts carried by tolerant mice (§3·3), or by mice in which the primary immune response had been temporarily suppressed by injections of trypan blue (§3·2). Moreover, although lymphoid cells are known to be vulnerable to the action of cytotoxic antisera in vitro , high doses of hyperimmune antisera transfused into tolerant mice were unable to destroy the surviving descendants of the lymphoid cells which had been injected at birth to induce the state of tolerance in the first instance (§3·3). The effect of high doses of antisera on skin homografts, if any, is to prolong their lives (‘enhancement’), but the effect is barely discernible. The enhancing action of antiserum was made most clearly apparent by its power to prevent the development or expression of sensi­tivity in CBA mice which had received injections of cell-free antigenic matter of A -strain origin (§4). CBA mice which received A -strain antigen plus 1·0 ml. hyperimmune anti- A serum rejected A -strain skin grafts more slowly than mice which had received normal serum instead of immune serum. It is not likely that the antiserum acted directly upon the antigen, first because the antiserum was just as effective when injected as late as 3 days after the sensitizing dose of antigen (§4·1), and secondly, because the sensitizing power of antigen was not perceptibly reduced by exposure to the same quantity of antiserum in vitro before in­jection (§4·2). Nor is antiserum likely to have acted by coating (or otherwise lowering the vulnerability of) the cells of the skin graft used to measure the prevailing degree of sensi­tivity, for passive transfusions of antiserum did not lower the sensitivity of pre sensitized mice (§5). Antiserum probably affects the process of sensitization itself, acting by a ‘central’ inhibition of unknown character rather than by obstructing the afferent or efferent pathways of response (§7·2). Two elementary phenomena relating to the duration and degree of the sensitivity produced by homografts require an explanation, ( a ) The sensitivity produced in mice by a single skin homograft is still clearly in force 240 days after its transplantation, but the sensitivity produced by a single intraperitoneal injection of 0·125 to 5·0 million lymphoid cells declines to a small fraction of its original value within 20 days (§ 6·1). ( b ) The repeated injection of high doses of foreign lymphoid cells, though provoking the formation of high titres of haemag-glutinins and haemolysins, does not raise the sensitivity of their recipients: a CBA mouse which has received six weekly intraperitoneal injections of 25 to 125 million A -strain lymphoid cells rejects an A -strain skin graft less promptly than a CBA mouse which has received a single injection of 5 million A -strain lymphoid cells 3 days beforehand (§ 6·2). No important fraction of this decline of sensitivity can be attributed to a ‘graft-versus-host’ reaction (§6·2). There is a temptation to believe that the decline of sensitivity that follows or accom­panies single or repeated injections of lymphoid cells is a ‘self enhancement’ attributable to the formation of humoral antibodies, but there are objections to this view, and a number of alternative possibilities are discussed (§§7·3, 7·4).
Article
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Tumor relapse is the primary cause of mortality in patients with hematologic cancers following autologous hematopoietic stem cell transplantation (HSCT). Vaccination early after HSCT can exploit both the state of lymphopenia and minimal residual disease for generating antitumor immunity. Here, multiple vaccinations using lymphoma cells engineered to secrete heat shock protein fusion gp96-Ig within 2 weeks of T cell-replete syngeneic HSCT led to cross-presentation and increased survival of lymphoma-bearing mice. To enhance vaccine efficacy, interleukin (IL)-2 was directed to predominantly memory phenotype CD8(+) T lymphocytes and natural killer (NK) cells via administration bound to anti-IL-2 monoclonal antibody clone S4B6 (IL-2S4B6). Combination therapy with gp96-Ig vaccination and coordinated infusions of IL-2S4B6 resulted in marked prolongation of survival, which directly correlated with ~500% increase in effector CD8(+) T-cell numbers. Notably, this dual regimen elicited large increases in both donor CD8(+) T and NK cells, but not CD4(+) T lymphocytes; the former 2 populations are essential for both vaccine efficacy and protection against opportunistic infections after HSCT. Indeed, IL-2S4B6-treated HSCT recipients infected with Listeria monocytogenes exhibited decreased bacterial levels. These preclinical studies validate a new strategy particularly well suited to the post-HSCT environment, which may augment adaptive and innate immune function in patients with malignant disease receiving autologous HSCT.
Article
An improved technique for specific adsorption of cytotoxic T lymphocytes on corresponding target cell monolayers is described. The adsorption process is accelerated by a 5-min centrifugation and results in 70–90% specific depletion of cytotoxic cells. The whole procedure requires 20 min.
Article
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Lineage-determining transcription factors (LD-TFs) drive the differentiation of progenitor cells into a specific lineage. In CD4+ T cells, T-bet dictates differentiation of the TH1 lineage, whereas GATA3 drives differentiation of the alternative TH2 lineage. However, LD-TFs, including T-bet and GATA3, are frequently co-expressed but how this affects LD-TF function is not known. By expressing T-bet and GATA3 separately or together in mouse T cells, we show that T-bet sequesters GATA3 at its target sites, thereby removing GATA3 from TH2 genes. This redistribution of GATA3 is independent of GATA3 DNA binding activity and is instead mediated by the T-bet DNA binding domain, which interacts with the GATA3 DNA binding domain and changes GATA3′s sequence binding preference. This mechanism allows T-bet to drive the TH1 gene expression program in the presence of GATA3. We propose that redistribution of one LD-TF by another may be a common mechanism that could explain how specific cell fate choices can be made even in the presence of other transcription factors driving alternative differentiation pathways.
Article
Human mucin 1 (MUC1) is a highly glycosylated transmembrane glycoprotein that is expressed on the luminal surface of ductal epithelial cells. Human adenocarcinomas overexpress MUC1 as a tumor-associated antigen (TAA) that presents to the immune system peptide epitopes and glycopeptide epitopes with tumor specific carbohydrates, such as mono- and disaccharides known as Tn, sialyl-Tn, and TF antigens. Studies in MUC1 transgenic (MUC1-Tg) mice have indicated that, compared to transgene negative wild-type (WT) mice, the MUC1-Tg immune system maintains a certain level of tolerance to the MUC1 peptide, reflected most notably in decreased CD4 T cell help. We made a novel observation that in contrast to suppressed responses to the MUC1-peptide vaccine, vaccination of MUC1-Tg mice with tumor-associated MUC1 glycopeptide resulted in effective anti-MUC1 immunity similar to that elicited in WT mice. We hypothesized that the tumor-associated glycopeptides were seen as foreign and therefore not subject to tolerance. To study CD4 T cell responses to MUC1 glycopeptides we generated glycopeptide GST(GalNAc;Tn)A specific, MHC-Class II restricted CD4 T cell hybridoma, RF6. We cloned the RF6 TCR (RFT; Vá4.1Já16-Vâ15Jâ1.3) and generated TCR transgenic mice RFT-Tg. Using the RFT-Tg mice and the previously generated MUC1 peptide-specific TCR transgenic VFT-Tg mice, we show that peptide-specific VFT CD4 T cells transferred to MUC1-Tg mice are suppressed through mechanisms of peripheral tolerance, which are not induced against MUC1-glycopeptide specific RFT CD4 T cells. We show that peptide-specific CD4 T cells are transiently activated upon transfer into MUC1-Tg mice, suggesting that MUC1-peptide epitope is presented in the periphery in healthy mice as well as in tumor bearing mice. In contrast, MUC1-glycopeptide epitopes are tumor specific and thus treated as foreign antigens in MUC1-Tg mice, resulting in effective activation of glycopeptide-specific CD4 T cells. Simultaneous activation of glycopeptide-specific T cells can break tolerance of peptide-specific T cells. Our findings with MUC1 apply to other TAA that contain some epitopes that are "self" and subject to self tolerance and other epitopes that are tumor specific ("foreign") and not affected by tolerance. Understanding this distinction is very important in the development of effective and safe cancer vaccines.
Article
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We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nefmut exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nefmut fused with HPV E7. In this way, we predicted that the expression of the Nefmut/E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host) engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nefmut/green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nefmut/E7 developed a CD8⁺ T-cell immune response against both Nef and E7. Conversely, no CD8⁺ T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA-injected mice was formally demonstrated by the E7-specific CD8⁺ T-cell immune response we detected in mice inoculated with exosomes isolated from plasma of mice inoculated with the Nefmut/E7 vector. Finally, we provide evidence that the injection of Nefmut/E7 DNA led to the generation of effective antigen-specific cytotoxic T lymphocytes whose activity was likely part of the potent, therapeutic antitumor effect we observed in mice implanted with TC-1 tumor cells. In summary, we established a novel method to generate immunogenic exosomes in vivo by the intramuscular inoculation of DNA vectors expressing the exosome-anchoring protein Nefmut and its derivatives.
Article
An electrophoretic mobility shift assay was used to characterize interactions of nuclear proteins with a DNA segment in the enhancer element of the leukemogenic murine retrovirus SL3-3. Mutation of a DNA sequence of the 5'-TGTGG-3' type decreased transcription in vivo specifically in T-lymphocyte cell lines. Extracts of nuclei from different T-lymphocyte cell lines or cells from lymphoid organs resulted in much higher amounts of complexes in vitro with this DNA sequence than did extracts from other cell lines or organs tested. Differences were also found in the sets of complexes obtained with extracts from the different types of cells. The DNA sequence specificities of the different SL3-3 enhancer factor 1 (SEF1) protein complexes were found to be distinct from those of several other previously identified DNA motifs of the TGTGG type because of differences in several nucleotides critical for binding and because these other DNA motifs could not compete with the identified DNA sequence for binding of SEF1. Limited treatment with several different proteases cleaved the SEF1 proteins such that their DNA-binding domain(s) remained and created complexes with decreased and nondistinguishable electrophoretic mobility shifts and with new properties. These results indicate that the SEF1 proteins have a structure with a flexible and relatively vulnerable hinge region linking a DNA-binding domain(s) to a more variable domain(s) with other functions. We suggest that the binding of SEF1 is an essential factor for the T-cell tropism of SL3-3 and the ability of this virus to cause T-cell lymphomas.
Article
Recent studies in both mice and humans have suggested that gut microbiota could modulate tumor responsiveness to chemo- or immunotherapies. However, the underlying mechanism is not clear yet. Here, we found that gut microbial metabolites, especially butyrate, could promote the efficacy of oxaliplatin by modulating CD8⁺ T cell function in the tumor microenvironment. Butyrate treatment directly boosted the antitumor cytotoxic CD8⁺ T cell responses both in vitro and in vivo in an ID2-dependent manner by promoting the IL-12 signaling pathway. In humans, the oxaliplatin responder cancer patients exhibited a higher amount of serum butyrate than did non-responders, which could also increase ID2 expression and function of human CD8⁺ T cells. Together, our findings suggest that the gut microbial metabolite butyrate could promote antitumor therapeutic efficacy through the ID2-dependent regulation of CD8⁺ T cell immunity, indicating that gut microbial metabolites could be effective as a part of cancer therapy.
Chapter
Of all those who studied the rejection of tumors or normal tissues, it was J.B. Murphy who had the clearest understanding of the direct causal relationship between allograft rejection and the presence of host lymphocytes in the graft. He found that resistance to allogeneic tumors, induced by previous inoculation of defibrinated blood from the tumor donors, was accompanied by a definite lymphoid crisis in the blood (i.e., lymphocytosis). He observed that lymphocytes were a necessary factor in the immunity processes. He quoted in support of Apolant's finding that previous splenectomy interfered with the development of immunity and mentioned that a role for antibodies in cancer immunity had not been found. The development in the understanding of histocompatibility antigens is considered in the chapter. Dempster, a transplant surgeon, made some contributions to the understanding of kidney-allograft rejection in dogs and in patients and wrote several reviews on the allograft reaction, and his work is discussed in the chapter. The discovery of the genetic basis of the histocompatibility antigens and of alloreactivity is elaborated in the chapter as well.
Thesis
Many tumour cells express high levels of proteins which are present in normal cells at lower levels. Such proteins might be targets for immunotherapy if lymphocytes with specificity for these proteins are present in the repertoire and can discriminate between cells expressing high or low levels of the target protein. The first step in exploring the possibility of targeting immunotherapy to normal self proteins is therefore to determine whether lymphocytes specific for molecules overexpressed in tumours exist and can be activated to recognize specifically cells expressing high levels of the proteins. It was possible to stimulate murine cyclin D1 or mdm2 specific cytotoxic T lymphocytes (CTL) by in vivo priming of mice with recombinant vaccinia virus expressing the proteins. The in vitro conditions used for restimulation of in vivo primed CTL were found to have a profound effect on the magnitude of the CTL response observed in vitro. In addition, in vitro priming of CTL from naïve spleens with peptide pulsed dendritic cells identified a mdm2 derived peptide, mdp441, which could stimulate CTL capable of recognising endogenously processed mdm2. These peptide specific CTL were of high avidity in contrast to most of the peptide specific CTL stimulated with other self peptides. The mdp441 peptide was not recognised by mdm2 specific CTL generated by in vivo immunisation with mdm2 protein. The results show that tolerance to self proteins is not absolute and that it is possible to stimulate CTL to recognize endogenously processed self protein either by in vivo immunisation with recombinant vaccinia virus expressing the self protein or by in vitro priming of naive responder cells with peptide pulsed antigen presenting cells. These results have implications for immunotherapy of human cancers.
Article
Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Hypoxia is a common feature of solid tumors, and cells adapt by upregulating the transcription factor HIF-1α. Here, we defined the transcriptional landscape of mouse tumor-infiltrating natural killer (NK) cells by using single-cell RNA sequencing. Conditional deletion of Hif1a in NK cells resulted in reduced tumor growth, elevated expression of activation markers, effector molecules, and an enriched NF-κB pathway in tumor-infiltrating NK cells. Interleukin-18 (IL-18) from myeloid cells was required for NF-κB activation and the enhanced anti-tumor activity of Hif1a−/− NK cells. Extended culture with an HIF-1α inhibitor increased human NK cell responses. Low HIF1A expression was associated with high expression of IFNG in human tumor-infiltrating NK cells, and an enriched NK-IL18-IFNG signature in solid tumors correlated with increased overall patient survival. Thus, inhibition of HIF-1α unleashes NK cell anti-tumor activity and could be exploited for cancer therapy.
Thesis
The possibility of inducing cytotoxic T lymphocytes (CTL) to mutant or normal Ras has been investigated using recombinant vaccinia viruses expressing these proteins. Mutant Ras or over expression of Ras is frequently associated with human malignancies, being implicated in 40% of colorectal cancers and 95% of pancreatic cancers. Oncogenic activation occurs by single point mutation of amino acid 12, 13 or 61. The structurally mutated or over expressed Ras oncogene has the potential of producing novel peptide antigens, which if presented on the cell surface by class I MHC molecules may be recognized by cytotoxic T cells. These may serve as targets for immunotherapy of certain cancers. Recombinant vaccinia viruses expressing normal (Vac-Ras) or mutant (Vac-Ras6l) Ras were constructed. Vac-Ras61 immunized C57Bl/10 mice (H-2b) developed Ras61-specific CTL which recognized normal Ras inefficiently. CTL isolated from Vac-Ras immune mice showed the opposite specificity. Endogenous levels of Ras expression were insufficient for lysis. Synthetic Ras peptides with an H-2Kb motif were identified. Peptide binding of MHC class I was demonstrated using the H-2b derived RMA-S mutant thymoma cell line in which peptide induced class I stabilisation was detected by immunofluorescence. One CTL epitope mapped to amino acids 60-67 and residue 61 was critical for T cell recognition. Anti-Ras61 CTL recognised peptide 60-67 containing mutant residue 61, while anti-Ras CTL recognised wild type 60-67. A second epitope mapped to residues 152-159 of Ras and was recognised equally well by CTL raised to normal and mutant Ras. These peptides could stimulate in vitro proliferation of specific CTL which recognised target cells pulsed with the appropriate peptides. The murine data suggest the possibility of exploiting Ras-specific CTL for targeted immunotherapy of certain human cancers.
Article
Canine B cell lymphoma is one of the most common hematopoietic neoplasms in veterinary medicine, and it is considered a relevant model for human diffuse large B cell lymphoma (DLBCL). Although the standard treatment consisting of multidrug chemotherapy is effective in most cases, treatment is often challenging because of relapse and drug resistance. The adoptive transfer of autologous T cells genetically modified to express a CD19‐specific chimeric antigen receptor (CD19 CAR‐T cells) has been shown to be highly effective in human B cell malignancies. However, there is no clinically available canine CAR‐T cell therapy. We generated canine second‐ and third‐generation CAR‐T cells by retroviral gene transduction. Optimization was performed to investigate effective viral transduction protocols and favorable culture conditions for canine CAR‐T cells. The RetroNectin®‐bound virus infection method resulted in more than 70% transduction efficiency. The effect of culture conditions on the phenotype of CAR‐T cells was evaluated by the expression of surface markers. In vitro cytotoxicity assays of target cells cultured with CD20 CAR‐transduced cells demonstrated that CD20 CAR‐T cells exhibit cytotoxicity against CD20‐expressing canine B cell lymphoma cells and canine CD20‐transduced murine cells, whereas no effect was observed against cells that lacked canine CD20 expression. Our study established virus‐based canine CAR‐T cell generation, providing fundamental data for a better understanding of canine adoptive T cell therapy. This article is protected by copyright. All rights reserved.
Article
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Monoterpenes are naturally occurring hydrocarbons composed of two units of isoprenes. They exhibit antioxidant activity to scavenge reactive oxygen species, such as hydroxyl radicals. We investigated the potential of monoterpenes such as thymol, linalool, and menthol to act as radioprotectants. The proliferation of EL4 cells, a mouse lymphoma cell line, treated with linalool at a concentration of 500 μM or more was not affected by X-ray irradiation. Plasmid-nicking assay performed using formamidopyrimidine-DNA glycosylase showed that linalool prevented single strand breaks and oxidized purines on pUC19 plasmid DNA. These findings indicate that linalool has the ability to scavenge reactive oxygen species and is a potential radioprotector.
Article
The history of the H-2 complex stems from the studies of tumor immunology and represents the foundations of a distinct modern discipline—the immunogenetics of histocompatibility.
Chapter
Host’s immunity against tumor cells can be classified according to two distinct functions, i.e. elicitable responses, evoked by tumor-associated antigens, and natural resistance (NR) not requiring previous exposure to antigenic determinants.
Article
We investigated the generation of CD8 cytotoxic T-lymphocytes (CTL) that recognized a dominant pRL1a peptide bound to H-2L(d) molecule on RL male 1 leukemia in spleen cells from RL male 1-bearing, syngeneic BALB/c, semiallogeneic CB6F(1) and allogeneic B6 mice by repetitive in vitro stimulation with RL male I tumor. CD8 T cells in cultures were also analyzed by H-2L(d)/pRL1a tetramer staining. We showed that pRL1a-specific CTL were more efficiently generated in spleen cells from RL male 1-bearing high responder CB6F(1) mice than in low responder BALB/c mice, and this correlated well with the occurrence of H-2L(d)/pRL1a tetramer binding CD8 T cells. Furthermore, we showed that in spleen cells from RL male 1-bearing allogeneic B6 mice, H-2L(d)/pRL1a complex specific CD8 T cells were present at a significant frequency. H-2L(d)/pRL1a recognizing B6 CTL but not BALB/c or CB6F(1) CTL gradually lost CD8 expression on their surface by multiplication of in vitro stimulation.
Article
Upon infection with the Moloney murine sarcoma virus-murine leukemia virus (MuLV) complex, H-2(b) C57BL/6 (B6) mice respond with a class I D-b-restricted cytotoxic T-lymphocyte (CTL) response, which protects against virus-induced tumorigenesis. In the B6-derived D-b mutant B6.CH-2(bm13) (bm13) strain, part of the class I D-b antigen-presenting groove is shaped by a class I K-b-encoded sequence. Like B6 mice, bm13 mice reject Moloney virus-induced tumors, but the protective CTL response is K-b restricted. In this study we show enhanced levels of Moloney MuLV-specific CTLp with a restriction for K-b in bm13 mice. Through the use of CTL clones from Moloney virus-immunized bm13 mice, the class I K-b-presented CTL epitope was identified. The epitope is located in the Moloney virus gp70 envelope protein region {Moloney envelope, amino acids 189 to 196 [Mol env (189-196)]}, SSWDFITV and has the K-b allele-specific binding motif. The D-bm13 molecule does not present the env(189 to 196) epitope to K-b-restricted bm13 CTL. In B6 mice, Mol env(189-196)-specific CTL could be induced by peptide vaccination. B6 mice thus have CTL precursors specific for this epitope but at considerably lower levels than do bm13 mice. We hypothesize that additional positive selection K-b-restricted CTL on the D-bm13 molecule in bm13 mice explains this difference in precursor frequencies. We examined related strains of MuLV for the presence of Mol env(189-196) sequence equivalents. Rauscher, Friend, and AKV MuLV-encoded Mol env(189-196) epitope equivalents were properly recognized in cytotoxicity assays, both as synthetic and as endogenously expressed (Rauscher MuLV) peptides. In contrast, the mink cell focus-forming virus MuLV-encoded epitope equivalent, lacking a K-b anchor residue, was not presented for CTL recognition and hence can be excluded as an important CTL epitope for mink cell focus-forming viruses.
Article
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Here we report two monoclonal antibodies, KP14/1 and KP15/11, produced by fusion of lymph node cells from a mouse transgenic for a soluble analog of the mouse MHC class I molecule H-2Dd that had been immunized with purified soluble H-2Dd loaded in vitro with the decamer antigenic peptide, p18-110, derived from the envelope gp120 of the IIIB isolate of HIV. These antibodies specifically bind E-2Dd complexed with p18-110, but do not bind HLA-A2.1 assembled with the same peptide. They fail to bind H-2Dd complexed with peptides unrelated to p18-110. To understand the structural basis for the recognition of MHC/peptide complexes by such antibodies, we made complexes from H-2Dd and each of a panel of peptides related to p18-110. The antibodies bound this panel of MHC/peptide complexes with similar but not identical patterns. The pattern was similar to but distinct from that of a T cell receptor derived from an H-2Dd-restricted p18-110 specific T cell hybridoma that we have recently characterized. Scrutiny of the encoded amino acid sequence of the two antibodies as compared to the T cell receptor indicates some regions that are conserved among the three molecules. The two monoclonal antibodies utilized the identical VH gene segment which remarkably had a stretch of amino acid identity with the TCR we have studied. We conclude that antibodies with specificity similar to that of T cell receptors can be isolated and that these leave a similar molecular footprint on the MHC/peptide complex. Thus the plasticity of the B cell receptor repertoire allows antibodies to effectively mimic αβ T cell receptors.
Article
A portion of the human cellular homolog of v-rel, the transforming gene of the leukemogenic retrovirus reticuloendotheliosis virus, strain T, was used to survey RNAs from several mouse tissues, selected lymphocyte populations, and hematopoietic cell lines for c-rel expression. Relatively high levels of a high-molecular-weight transcript were observed in peripheral B and T cells, whereas lower levels were detectable in functionally immature thymocytes. These results suggested that, unlike c-myb and c-ets, the c-rel proto-oncogene plays a role in later stages of lymphocyte differentiation.
Book
This volume is essential for geneticists, molecular biologists, biochemists, and medical doctors interested in the use of mouse models in cancer research. Recent genome studies, together with refined genetic engineering techniques, have greatly increased the value of using mice for research on cancer and other human disorders. The chapters of this book will support scientists in choosing the most suitable mouse models for their research questions. The book provides detailed methodological information for genetic or chemical induction of different types of cancer, histomorphometric cancer analysis, and in vivo imaging, as well as protocols to investigate oncogene addiction, immune surveillance, and hallmarks of cancer such as angiogenesis or metastasis. Four review-like articles provide background information on mouse technologies and histopathologic differences between mouse and human cancers. The mouse models described in individual chapters will fuel the understanding of cancer initiation, immune system roles, tumor angiogenesis, invasion, metastasis, and the relevance of molecular diversity observed among human cancers. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and resourceful, Mouse Models of Cancers: Methods and Protocols, is a valuable laboratory resource for all researchers, from the graduate level upwards, who study cancer and new possibilities for its treatment. © Springer Science+Business Media New York 2015. All rights are reserved.
Article
Dendritic cells (DC) are a system of antigen-presenting cells specialized in interaction with T cells. Recently it has been reported that DC can produce CC (β) chemokines that attract T cells. In this study we isolated mouse fractalkine and macrophage-derived chemokine (MDC) belonging to CX3C (δ) and CC chemokine families, respectively, from bone marrow-derived mature DC. While expression of fractalkine, which has so far been only examined in the brain and in vitro endothelial cells so far, was rather ubiquitous, MDC, which has been reported to be synthesized by macrophages and DC, was expressed specifically in the thymus and lymphnode. This is the first report that indicates fractalkine expression by DC. Expression of fractalkine and MDC mRNA increased with maturation of DC during in vitro culture of bone marrow cells. Spleen- and epidermis-derived mature DC in culture also expressed these chemokines. Furthermore, their expression was detected selectively by Northern hybridization in CD11c+ B220– DC freshly purified from lymph nodes, and in large stellate cells in the lymph node T cell areas by in situ hybridization. Conditioned media of 293T cells transfected with these chemokine cDNA were chemotactic to Con A-activated splenic T cells as well as the mouse T cell line EL4. In conclusion, while fractalkine and MDC belong to different families of chemokines, both may be involved in recruitment of T cells for interaction with mature DC in the immune response.
Article
The advantages presented by the use of the comparatively homogeneous free tumor cell suspension of the Ehrlich ascites tumor for quantitative studies on the growth and chemical composition of tumor cells and for chemotherapeutic experiments have led to the present investigation of a representative array of 26 histologically and genetically different mouse tumors which were tested for their capacity of reaching a condition similar to the Ehrlich ascites tumor. This has been found to be possible in a number of cases. Dissociated solid tumor masses were intraperitoneally inoculated into the corresponding mouse strain. Tumor growth on the peritoneum, accompanied by exudate formation, resulted in 22 out of 26 cases. The resulting exudates, which, in the case of all carcinomas and most sarcomas tested, contained very few tumor cells, transmitted the tumors upon further intraperitoneal injection in 18 instances. The tumors which developed were now regularly provoking exudate formation, and on subsequent generations the exudates have always been transplanted intraperitoneally. In the case of all carcinomas and in 2 out of 3 sarcomas tested which have passed over 10 such “fluid transplant generations” (FTG), a gradual increase in the frequency of tumor cells in the ascitic fluids (examined around median survival time) was occurring after a certain
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