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El Khawanky, Nadia, Amy Hughes, Wenbo Yu, Sanaz Taromi, Jade Clarson, Angel F. Lopez, Michael P. Brown, et al. "Azacytidine Sensitizes AML Cells for Effective Elimination By CD123 CAR T-Cells." Blood 134, Supplement_1 (November 13, 2019): 3904. http://dx.doi.org/10.1182/blood-2019-124684.
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Chimeric antigen receptor T-cells (CAR Tc) have yielded impressive remission rates in treatment-refractory B-cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in the first FDA approved CAR Tc therapies, Kymriah and Yescarta. However, the translation of these results for other cancer entities remains a challenge. Pre-clinical studies using second-generation CAR Tc against the interleukin-3 receptor alpha chain (CD123) engendered strong anti-leukemic activity. CD123 CAR Tc clinical studies resulted in transient responses, or complete remission but at the expense of on-target off-tumor toxicities. Our studies employing third-generation anti-CD123 CAR Tc demonstrate strong anti-leukemic activity with no adverse effects in vivo. However, the leukemia was not completely eradicated. Combining anti-CD123 CAR Tc with DNA hypomethylating (HMA) agents may enhance the anti-leukemic effect and survival. HMAs such as azacytidine (Aza) activate key epigenetically silenced pathways in AML cells, inhibiting cell proliferation while enhancing cell immunogenicity. We hypothesized that Aza will increase the expression of CD123 on AML cells resulting in long-term disease eradication by anti-CD123 CAR Tc. The anti-leukemic efficacy, survival advantage, safety and feasibility of the combination treatment with Aza and anti-CD123 CAR Tc were evaluated in vivo. HL-60 (CD123med), MLL-2 (CD123lo), MOLM-13 (CD123hi), primary de novo and relapsed/refractory (r/r) AML cells were cultured for 0-8 days in the presence of Aza (0µM-5µM) and analysed for their CD123 expression by flow cytometry, quantitative western blot and RNAseq. The anti-CD123 CAR was constructed with the humanized CSL362-based ScFv and the CD28-OX40-CD3ζ signaling domain, encoded in a third-generation lentiviral vector and expressed in CD3+ Tc from healthy donors. Rag2γc-/- mice (n=12-16/ group) were engrafted with 1x105 MOLM13/ffLuc AML cells and treated with PBS, 5x106 Non-transduced (NTD) Tc orCAR Tc, 4x 2.5mg/kg Aza, or 5x106 CAR Tc following 4x Aza (2.5mg/kg). Leukemic burden was assessed weekly by bioluminescence imaging. Tc activity and immunophenotyping was performed using flow cytometry at day 35 post engraftment, and survival was monitored. HL-60, MLL-2 and MOLM-13 cells showed significant increases in HLA-DR, PD-L1, STAT1 and IRF7 expression, as well as CD123 when exposed to Aza (Fig 1A,B). Interestingly, the increased effect was seen from day one regardless of concentration. This was similarly reflected in AML patient cells. Aza treatment also arrested cell proliferation and decreased viability in both cell lines and patient cells suggesting Aza can aid in the anti-leukemic effect. Rag2γc-/- mice engrafted with MOLM-13 and treated with Aza and CD123 CAR Tc demonstrated suppressed growth, and eradication of MOLM-13 cells compared to mice treated with CD123 CAR Tc or Aza alone. Additionally, a significant decrease in residual CD123+ cells in the bone marrow (BM) of dual treated mice was seen (Fig 1C). A higher frequency of residual CD8+ T-cells in the BM, and CD4+ Tc in the peripheral blood (PB) and BM of dual treated mice was observed compared to CAR Tc only treated mice. Most prominently, we found a significantly higher mean number of stem cell-like and central memory CD8+ Tc in the BM of dual treated mice (232 cells/µl and 208cells/µl, respectively) compared to the CAR Tc only group (55 cells/µl and 23 cells/µl, respectively). Assessment of immune checkpoint markers on residual CAR Tc of dual treated mice revealed significantly decreased levels of CTLA-4, PD-1 and TIM-3 in the BM, and CTLA-4 in the PB compared to the CAR Tc only group. While CAR Tc treatment alone demonstrated a survival advantage compared to PBS, NTD or Aza treated mice, Aza and CAR Tc treatment had a significantly higher survival rate compared to the CAR Tc only group (92% vs. 46% at day 50, p<.01). Our findings indicate that Aza increases immunogenicity and augments the cell surface expression of CD123 on AML cells, allowing enhanced recognition and elimination of malignant cells by CD123 CAR Tc. This is the first demonstration that HMAs and CAR Tc immunotherapy can be used synergistically to treat AML. Considering HMAs are currently under clinical investigation in AML, our data encourage further clinical evaluation of this dual treatment in r/r AML, including high-risk patients that are chemotherapy or allogeneic transplantation ineligible. Disclosures Hughes: Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Yong:Novartis: Honoraria, Research Funding; Celgene: Research Funding; BMS: Honoraria, Research Funding.
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Kos, F. J., and E. G. Engleman. "Requirement for natural killer cells in the induction of cytotoxic T cells." Journal of Immunology 155, no. 2 (July 15, 1995): 578–84. http://dx.doi.org/10.4049/jimmunol.155.2.578.
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Abstract Cell-mediated immunity involves the participation of both regulatory and cytotoxic cells. The conversion of precursors to effector CD8+ cytotoxic T (Tc) cells requires cell-cell collaboration in which CD4+ T cells are traditionally viewed as helper cells. An in vitro system was used here to demonstrate that the generation of human alloantigen-specific CD8+ Tc cells requires the participation of CD3-CD16+CD56+ NK cells but not CD4+ T helper cells. Depletion of NK cells from responders abolished the induction of alloantigen-specific Tc cells in mixed lymphocyte cultures (MLC). Purified CD5+CD8+ T cells stimulated with alloantigen proliferated but did not differentiate into fully functional effector Tc cells. Coculture of responder CD5+CD8+ T cells with NK cells promoted the conversion of CD8+ Tc cell precursors (pTc) into effector Tc cells. Anti-CD56 mAbs blocked Tc cell induction in MLC, suggesting a role for CD56 molecules expressed on NK cells in either alloantigen recognition or delivery of accessory signals to pTc cells. These findings suggest a novel critical link between the natural and specific immune responses.
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Kim, Sung Man, Eun Ju Lee, Hye Sook Jung, Na Han, You Jeong Kim, Tae Kyoon Kim, Tae Nyun Kim, et al. "Co-Culture of α TC-6 Cells and β TC-1 Cells: Morphology and Function." Endocrinology and Metabolism 30, no. 1 (2015): 92. http://dx.doi.org/10.3803/enm.2015.30.1.92.
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Inoue, Tsuyoshi, and Keiji Imoto. "Feedforward Inhibitory Connections From Multiple Thalamic Cells to Multiple Regular-Spiking Cells in Layer 4 of the Somatosensory Cortex." Journal of Neurophysiology 96, no. 4 (October 2006): 1746–54. http://dx.doi.org/10.1152/jn.00301.2006.
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Thalamocortical (TC) cells in the ventrobasal thalamus make direct excitatory connections with regular-spiking (RS) cells in layer 4 of the somatosensory cortex, but also make disynaptic feedforward inhibitory connections with the RS cells by layer 4 fast-spiking (FS) cells. In this study, we investigated connection rules of the feedforward inhibitory circuit from multiple TC cells to multiple RS cells, at the level of synaptic potentials. Using thalamocortical brain slices of young mice (postnatal days 12–16), we made simultaneous patch-clamp recordings from three adjacent cortical cells (two RS cells and one FS cell), combined with minimal stimulation of presumed single TC fibers. We found that nearly all (97%) of TC fibers, which generated excitatory inputs onto RS cells, also generated divergent excitatory inputs onto adjacent FS cells. Some 44% of TC fibers generated divergent excitatory inputs onto adjacent pairs of RS cells. We then combined the triple patch-clamp recording with multisite (two to three) minimal stimulation of single TC fibers and found that 86% of FS cells received convergent inputs from all of the stimulated TC fibers. We also found that 68% of FS cells generated divergent inhibitory inputs onto adjacent pairs of RS cells. The results indicate that spikes in TC cells, which excite RS cells, also excite adjacent FS cells with high fidelity. The results also indicate that FS cells receive convergent excitatory inputs from multiple TC cells and then send divergent inhibitory outputs to multiple RS cells.
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Reuter, Audrey, Chloé Virolle, Kelly Goldlust, Annick Berne-Dedieu, Sophie Nolivos, and Christian Lesterlin. "Direct visualisation of drug-efflux in live Escherichia coli cells." FEMS Microbiology Reviews 44, no. 6 (August 6, 2020): 782–92. http://dx.doi.org/10.1093/femsre/fuaa031.
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ABSTRACT Drug-efflux by pump proteins is one of the major mechanisms of antibiotic resistance in bacteria. Here, we use quantitative fluorescence microscopy to investigate the real-time dynamics of drug accumulation and efflux in live E. coli cells. We visualize simultaneously the intrinsically fluorescent protein-synthesis inhibitor tetracycline (Tc) and the fluorescently labelled Tc-specific efflux pump, TetA. We show that Tc penetrates the cells within minutes and accumulates to stable intracellular concentration after ∼20 min. The final level of drug accumulation reflects the balance between Tc-uptake by the cells and Tc-efflux by pump proteins. In wild-type Tc-sensitive cells, drug accumulation is significantly limited by the activity of the multidrug efflux pump, AcrAB-TolC. Tc-resistance wild-type cells carrying a plasmid-borne Tn10 transposon contain variable amounts of TetA protein, produced under steady-state repression by the TetR repressor. TetA content heterogeneity determines the cells’ initial ability to efflux Tc. Yet, efflux remains partial until the synthesis of additional TetA pumps allows for Tc-efflux activity to surpass Tc-uptake. Cells overproducing TetA no longer accumulate Tc and become resistant to high concentrations of the drug. This work uncovers the dynamic balance between drug entry, protein-synthesis inhibition, efflux-pump production, drug-efflux activity and drug-resistance levels.
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Carmel, R., SM Neely, and RB Jr Francis. "Human umbilical vein endothelial cells secrete transcobalamin II." Blood 75, no. 1 (January 1, 1990): 251–54. http://dx.doi.org/10.1182/blood.v75.1.251.251.
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Abstract Transcobalamin II (TC II) is essential for cellular uptake of cobalamin. However, the origin of this transport protein is controversial and many organ sources have been suggested. We studied human umbilical vein endothelial cells cultured in vitro. The cells contained TC II (2.3 pmol/10(8) cells) and released progressively increasing amounts of the protein into the surrounding medium during the 3-day incubation period. This release exceeded the starting intracellular content of TC II. In contrast, endothelial cells did not contain or elaborate R binder, the other major circulating binding protein for cobalamin, Cycloheximide inhibited the elaboration of TC II, suggesting that the endothelial cells synthesize the protein. Thrombin, which stimulates tissue plasminogen activator release, did not enhance TC II release, and neither did endotoxin or mellitin. However, thrombin did appear to partially protect TC II release from inhibition by cycloheximide. Among other cells studied, human fibroblasts also released TC II into the incubation medium, while K562 human leukemia cells, ARH-77 and HS Sultan human plasma cell lines, and Raji strain lymphoblasts did not. The data suggest that endothelial cells are an important source of the metabolically crucial TC II.
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Carmel, R., SM Neely, and RB Jr Francis. "Human umbilical vein endothelial cells secrete transcobalamin II." Blood 75, no. 1 (January 1, 1990): 251–54. http://dx.doi.org/10.1182/blood.v75.1.251.bloodjournal751251.
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Transcobalamin II (TC II) is essential for cellular uptake of cobalamin. However, the origin of this transport protein is controversial and many organ sources have been suggested. We studied human umbilical vein endothelial cells cultured in vitro. The cells contained TC II (2.3 pmol/10(8) cells) and released progressively increasing amounts of the protein into the surrounding medium during the 3-day incubation period. This release exceeded the starting intracellular content of TC II. In contrast, endothelial cells did not contain or elaborate R binder, the other major circulating binding protein for cobalamin, Cycloheximide inhibited the elaboration of TC II, suggesting that the endothelial cells synthesize the protein. Thrombin, which stimulates tissue plasminogen activator release, did not enhance TC II release, and neither did endotoxin or mellitin. However, thrombin did appear to partially protect TC II release from inhibition by cycloheximide. Among other cells studied, human fibroblasts also released TC II into the incubation medium, while K562 human leukemia cells, ARH-77 and HS Sultan human plasma cell lines, and Raji strain lymphoblasts did not. The data suggest that endothelial cells are an important source of the metabolically crucial TC II.
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Visciano, Carla, Nella Prevete, Federica Liotti, and Gianni Marone. "Tumor-Associated Mast Cells in Thyroid Cancer." International Journal of Endocrinology 2015 (2015): 1–8. http://dx.doi.org/10.1155/2015/705169.
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There is compelling evidence that the tumor microenvironment plays a major role in mediating aggressive features of cancer cells, including invasive capacity and resistance to conventional and novel therapies. Among the different cell populations that infiltrate cancer stroma, mast cells (MCs) can influence several aspects of tumor biology, including tumor development and progression, angiogenesis, lymphangiogenesis, and tissue remodelling. Thyroid cancer (TC), the most frequent neoplasia of the endocrine system, is characterized by a MC infiltrate, whose density correlates with extrathyroidal extension and invasiveness. Recent evidence suggests the occurrence of epithelial-to-mesenchymal transition (EMT) and stemness in human TC. The precise role of immune cells and their mediators responsible for these features in TC remains unknown. Here, we review the relevance of MC-derived mediators (e.g., the chemokines CXCL1/GRO-α, CXCL10/IP-10, and CXCL8/IL-8) in the context of TC. CXCL1/GRO-αand CXCL10/IP-10 appear to be involved in the stimulation of cell proliferation, while CXCL8/IL-8 participates in the acquisition of TC malignant traits through its ability to induce/enhance the EMT and stem-like features of TC cells. The inhibition of chemokine signaling may offer novel therapeutic approaches for the treatment of refractory forms of TC.
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Huang, Nick, Brandon Wyman, Emma Cravo, Thomas Winans, Gourav Choudhary, Zachary Oaks, Manuel Duarte, et al. "Rab4A inactivation in T cells blocks mTOR activation, pro-inflammatory lineage development, and disease pathogenesis in lupus-prone mice." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 115.6. http://dx.doi.org/10.4049/jimmunol.202.supp.115.6.
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Abstract Introduction Expression of Rab4A is increased in T cells of patients and mice with SLE. Overexpression of Rab4A forms a positive feedback loop with mTOR that can be blocked with therapeutic efficacy in SLE. To determine the impact of this gene on disease development, the triple-congenic lupus-prone Sle1.2.3 mouse strain (TC) have been backcrossed with C57Bl/6 wild-type (WT) mice that carry floxed Rab4AQ72L (TC-FL) alleles or lack Rab4A in T cells (TC-KO). Methods Proteinuria was assessed by the Bradford assay. Splenocytes were examined by flow cytometry. Autoantibody production was measured by ELISA. Results TC mice had increased proteinuria over age and sex-matched WT controls. Deletion of Rab4A in T cells reduced proteinuria ≥ 50% in female TC-KO mice relative to TC-FL mice at 21 or 40 weeks of age. Similar trends were noted in male mice. The production of antinuclear and antiphospholipid antibodies was reduced in TC-KO mice as compared to TC-FL and parental TC controls. Immunophenotyping unveiled a 45% depletion of CD4+ T cells and 50% expansion of CD8+ T cells in female TC-KO mice relative to TC-FL controls. CD38 expression was reduced on CD4+ T cells of TC-KO mice by 51% and 48% relative to TC and TC-FL controls. CD38 expression was also reduced on CD8+ T cells but not on CD19+ B cells. mTORC1 activity was reduced by 36% in CD4 T cells, but not in CD8 T cells or B cells of TC-KO mice. Along these lines, overexpression of Rab4A activated mTORC1, reduced expression of CD4, and increased expression of CD38 in Jurkat cells. Conclusion These findings reveal an opposite influence of Rab4A on endosomal recycling of CD4 and CD38 that facilitates mTORC1 activation and thus causes pro-inflammatory T-cell lineage specification and triggers autoimmunity in SLE.
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Destexhe, A., T. Bal, D. A. McCormick, and T. J. Sejnowski. "Ionic mechanisms underlying synchronized oscillations and propagating waves in a model of ferret thalamic slices." Journal of Neurophysiology 76, no. 3 (September 1, 1996): 2049–70. http://dx.doi.org/10.1152/jn.1996.76.3.2049.
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1. A network model of thalamocortical (TC) and thalamic reticular (RE) neurons was developed based on electrophysiological measurements in ferret thalamic slices. Single-compartment TC and RE cells included voltage- and calcium-sensitive currents described by Hodgkin-Huxley type of kinetics. Synaptic currents were modeled by kinetic models of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), gamma-aminobutyric acid-A (GABAA) and GABAB receptors. 2. The model reproduced successfully the characteristics of spindle and slow bicuculline-induced oscillations observed in vitro. The characteristics of these two types of oscillations depended on both the intrinsic properties of TC and RE cells and their pattern of interconnectivity. 3. The oscillations were organized by the reciprocal recruitment between TC and RE cells, due to their manual connectivity and bursting properties. TC cells elicited AMPA-mediated excitatory postsynaptic potentials (EPSPs) in RE cells, whereas RE cells elicited a mixture of GABAA and GABAB inhibitory postsynaptic potentials (IPSPs) in TC cells. Because of the presence of a T current, sufficiently strong EPSPs could elicit a burst in RE cells, and TC cells could generate a rebound burst following GABAergic IPSPs. Under these conditions, interaction between the TC and RE cells produced sustained oscillations. 4. In the absence of spontaneous oscillation in any cell, the TC-RE network remained quiescent. Spindle oscillations with a frequency of 9-11 Hz could be initiated by stimulation of either TC or RE neurons. A few spontaneously oscillating TC neurons recruited the entire network model into a "waxing-and waning" oscillation. These "initiator" cells could be an extremely small proportion of TC cells. 5. In intracellular recordings, TC cells display a reduced ability for burst firing after a sequence of bursts. The "waning" phase of spindles was reproduced in the network model by assuming an activity-dependent upregulation of Ih operating via a calcium-binding protein in TC cells, as shown previously in a two-cell model. 6. Following the global suppression of GABAA inhibition, the disinhibited RE cells produced prolonged burst discharges that elicited strong GABAB-mediated currents in TC cells. The enhancement of slow IPSPs in TC cells was also due to cooperativity in the activation of GABAB-mediated current. These slow IPSPs recruited TC and RE cells into slower waxing-and-waning oscillations (3-4 HZ) that were even more highly synchronized. 7. Local axonal arborization of the TC to RE and RE to TC projections allowed oscillations to propagate through the network. An oscillation starting at a single focus induced a propagating wavefront as more cells were recruited progressively. The waning of the oscillation also propagated due to upregulation of Ih in TC cells, leading to waves of spindle activity as observed in experiments. 8. The spatiotemporal properties of propagating waves in the model were highly dependent on the intrinsic properties of TC cells. The spatial pattern of spiking activity was markedly different for spindles compared with bicuculline-induced oscillations and depended on the rebound burst behavior of TC cells. The upregulation of Ih produced a refractory period so that colliding spindle waves merged into a single oscillation and extinguished. Finally, reducing the Ih conductance led to sustained oscillations. 9. Two key properties of cells in the thalamic network may account for the initiation, propagation, and termination of spindle oscillations, the activity-dependent upregulation of Ih in TC cells, and the localized axonal projections between TC and RE cells. In addition, the model predicts that a nonlinear stimulus dependency of GABAB responses accounts for the genesis of prolonged synchronized discharges following block of GABAA receptors.
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Namour, Farès, Jean-Luc Olivier, Idrissia Abdelmouttaleb, Charles Adjalla, Renée Debard, Colette Salvat, and Jean-Louis Guéant. "Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood." Blood 97, no. 4 (February 15, 2001): 1092–98. http://dx.doi.org/10.1182/blood.v97.4.1092.
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Abstract Transcobalamin (TC) is the plasma transporter that delivers vitamin B12 to cells. We have already reported that HT-29 and Caco-2 cells secrete different TC variants. HT-29 secretes 2 TC isoproteins (codon 259-Pro/Arg [259-P/R]), exhibiting unequal concentrations (TC 259-P > TC 259-R), and Caco-2 cells only secrete the phenotype 259-R. We investigated the relation between phenotypic and genetic TC polymorphism in HT-29 cells transfected with Caco-2 TC complementary DNA and in 159 healthy Caucasians. We found that codon 259-R is buried and, thus, the genetic polymorphism provides no explanation why the TCs from HT-29 and Caco-2 cells have different isoelectric points in nondenaturing isoelectric focusing (IEF). The newly translated TC in HT-29 cells from the Caco-2 complementary DNA recombinant plasmid had the same isoelectric point as the TC constitutively expressed in HT-29 cells, suggesting that TC phenotypic variability involves a specific cell folding of the protein. The codon 259 polymorphism was found to have a biallelic distribution: homozygotes P = 34.6%, heterozygotes R/P = 47.8%, and homozygotes R = 17.6%. In heterozygous samples, the IEF showed that the TC 259-P/TC 259-R ratio = 1.6. The blood apo-TC concentration of 259-P homozygous Caucasians was significantly higher than that of homozygous 259-R (P < .0001) and heterozygous (P < .0006) Caucasians. The heterozygotes 259-R/P had homocysteine concentration significantly higher than the homozygotes 259-R and 259-P (P = .02 and P = .01, respectively). In conclusion, TC codon-259 polymorphism affects TC plasma concentration and may interfere in vitamin B12cellular availability and homocysteine metabolism.
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Schwartz, L. B., A. M. Irani, K. Roller, M. C. Castells, and N. M. Schechter. "Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells." Journal of Immunology 138, no. 8 (April 15, 1987): 2611–15. http://dx.doi.org/10.4049/jimmunol.138.8.2611.
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Abstract Levels of histamine, chymase, and tryptase were assessed in preparations of dispersed human TC (tryptase+, chymase+) mast cells obtained from foreskin and of dispersed human T (tryptase+, chymase-) mast cells obtained from lung. Consistent with previous immunohistochemical results, extracts of T mast cells, the predominant mast cell type in lung (93% T and 7% TC mast cells), were deficient in human chymase (less than 0.3 microgram and 0.04 U/10(6) mast cells) but not tryptase (10.8 micrograms and 0.3 U/10(6) mast cells) by corresponding immunologic and enzymatic (suc-L-ala-ala-pro-phe-p-nitroanilide in the presence of aprotinin and tosyl-L-gly-pro-lys-p-nitroanilide in the presence of soybean trypsin inhibitor, respectively) assays. The minor presence of chymase activity in lung could be accounted for by the minor presence of lung TC mast cells. Extracts of TC mast cells, the predominant mast cell type (1% T and 99% TC mast cells) in foreskin, contained both proteases. However, TC mast cells from adult foreskin contained eightfold to 10-fold higher levels of chymase (4.5 micrograms and 1.01 U/10(6) mast cells) and twofold to threefold higher levels of tryptase (11.5 micrograms and 0.27 U/10(6) mast cells) than did TC mast cells from newborn foreskin (less than 0.6 microgram and 0.09 U of chymase and 35 micrograms and 0.62 U of tryptase/10(6) mast cells). In contrast, histamine levels were not significantly different in adult foreskin TC (1.9 microgram/10(6) mast cells), newborn foreskin TC (1.6 microgram/10(6) mast cells), and adult lung T (1.5 microgram/10(6) mast cells) mast cells. The relative ratio of each mediator in newborn foreskin mast cells to that in adult foreskin mast cells is highest for histamine, followed by tryptase and then chymase. Tryptase from TC and T mast cells had identical subunit compositions by Western blot analysis and similar apparent specific activities. This study extends the previously reported immunohistochemical distinction between human T and TC mast cells in tissue sections by direct quantitation of chymase and tryptase in dispersed preparations of T and TC mast cells.
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Zheng, Ying Yi. "Marginal zone B cells contribute to autoimmunity in the NZM2410 lupus prone mouse model. (159.13)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 159.13. http://dx.doi.org/10.4049/jimmunol.188.supp.159.13.
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Abstract Lupus is an autoimmune disease in which the affected individual generates antibodies (Abs) against nuclear antigens such as DNA. Our NZM2410-derived triple congenic (TC) model of lupus expresses 3 previously characterized lupus susceptibility loci. We have previously shown that the breach of follicular exclusion of TC MZB cells is associated with disease progression. The production of nuclear autoAbs occurred in tandem with a large influx of TC MZB cells in the follicles. We also found an expansion of MZB cells in TC mice as compared to non-autoimmune B6 mice that occurs prior to the production of anti-DNA autoAbs, further suggesting that MZB cells may contribute to autoimmunity. MZB cell expansion is associated with the upregulation of Notch2 signaling in these cells. Adoptive transfer of TC or B6 splenic B cells into B6.Rag1-/- recipients indicated no difference in the homeostatic expansion of MZB cells from either donor strain, whether the recipients were treated or not with CpG. However, CpG-treated recipients of TC B cells produced more anti-ssDNA IgM than recipients of B6 B cells. Our transfer results showed that although TC MZB cells do not expand more than B6 MZB cells in a lymphopenic host, TC splenic B cells produce higher level of anti-ssDNA IgM in response to CpG stimulation. Further characterization of the TC MZB cells will determine more precisely how they contribute to autoimmunity.
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Mueller, Antonia MS, Sumana Shashidhar, Janice Brown, and Judith A. Shizuru. "Adoptively Transferred Mature T Cells Exhibit Diminished Reactivity to Murine Cytomegalovirus Compared to Nascent Donor T Cells and Impair Functional Immune Recovery After Allogeneic Hematopoietic Cell Transplantation." Blood 116, no. 21 (November 19, 2010): 3732. http://dx.doi.org/10.1182/blood.v116.21.3732.3732.
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Abstract Abstract 3732 Infections due to impaired immune function are a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Adoptively transferred donor (do) T cells (TC) are thought to provide protective immunity, as TC-depleted grafts are associated with increased viral infections post HCT (pTX). TC-replete grafts, however, require immunosuppression to prevent GVHD, and can damage lymphoid organs causing additional immune dysfunction. Using an MHC-matched, minor antigen (miAg) mismatched GVHD model, we compared immune responses to murine cytomegalovirus (MCMV) in lethally irradiated BALB.B recipients infused with purified hematopoietic stem cells (HSC; cKit+Sca1+Thy1.1loLin−) or HSC+TC (subsets) from congenic C57BL/6 (B6) donors which could be distinguished based upon CD45 alleles. Hosts were infected with sublethal doses of MCMV at 2 or 8 weeks (w) pTX. 2w after infection, lymphoid organs were harvested to assess the immune function of: 1) transferred doTC from uninfected and pre-immunized donors, 2) HSC-derived doTC, and 3) residual host TC. Anti-MCMV CD8 TC were measured by M45 tetramer staining and an MCMV ELISPOT assay was used to measure IFNg production of FACS-separated doTC-, doHSC-, and host-derived spleen populations when exposed to the viral peptide. Mice given TC-replete allografts and infected at 2w pTX mounted significantly lower responses than control congenic- or HSC only recipients. In the latter group reactivity originated from residual host cells. Transplantation of TC from immunized donors did not improve the response of doTC transplanted into miAg-mismatched hosts. However, when HSC were supplemented with memory (CD62L+/−CD44+) CD8 TC from immunized donors, recipients of memory, but not of naive CD8 TC (CD62L+CD44−), mounted strong anti-MCMV responses in both assays. At 8w pTX recipients of pure HSC were mixed TC chimeras, while mice given HSC+TC were full donor chimeras with the majority of TC originating from transferred doTC. When mice were infected 8w pTX, anti-MCMV reactivity of CD8 TC was significantly greater in recipients of pure HSC, as compared to weak responses seen in recipients of HSC+TC. In recipients of pure HSC, both donor and host populations demonstrated strong reactivity, while mice given HSC+TC had weak responses of both doTC-, and doHSC-derived TC (Fig.1). When recipients of HSC alone or HSC + total TC (ToTC), naive CD8, memory CD8 or M45 tetramer+ CD8 TC were infected with a lethal dose of MCMV at 2w pTX, 50% of ToTC and 20% of HSC recipients died before day 40 pTX, while all mice given any of the CD8 subsets were protected from infection. 6w post MCMV infection organs of survivors were analyzed. Hepatic CD8 cells contained a median of <0.05% M45 tetramer reactive cells in ToTC-, <0.5% in HSC only- and naive CD8-, and 0.65% in memory CD8 recipients (p=0.03 compared to ToTC group). Strikingly, in mice given as few as 7,000 M45 tetramer+ cells, the proportion of M45-reactive cells reached a median 8.7% in the liver, and 4.3% in the spleen. In mice given ToTC, >90% of TC were doTC-derived with an effector memory phenotype. In contrast, recipients of naive, memory, or tetramer-sorted CD8 cells had TC originating from expanded doTC, doHSC, or residual host. Nascent doHSC-derived lymphopoiesis sustained a robust pool of naive TC - a sign of healthy immune reconstitution. Thus, our key findings are: 1) mature doTC do not maintain their full anti-viral potential when transplanted into miAg-disparate mice, even when obtained from pre-immunized donors; 2) residual host cells contribute substantially to protective immunity early pTX, but are eradicated by conventional TC-replete grafts; 3) HSC-derived TC arising in a healthy host are superior to those that develop and undergo selection in a GVHD-affected lymphoid system; and 4) low numbers of selected cell subsets, such as tetramer-sorted CMV-specific CD8 TC, expand dramatically in a lymphopenic environment and provide functional protection against infections. Our results challenge the conventional assumption that doTC in a hematopoietic allograft are required for optimal regeneration of the immune system. Rather, our studies suggest that long-term lymphoid function will greatly benefit from rigorous TC-depletion of the graft, and avoidance of GVHD. Moreover, co-transfer of small numbers of highly selected TC clones can provide effective antigen-specific protection. Disclosures: No relevant conflicts of interest to declare.
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Vannucchi, Maria Giuliana, and Chiara Traini. "Interstitial cells of Cajal and telocytes in the gut: twins, related or simply neighbor cells?" Biomolecular Concepts 7, no. 2 (May 1, 2016): 93–102. http://dx.doi.org/10.1515/bmc-2015-0034.
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AbstractIn the interstitium of the connective tissue several types of cells occur. The fibroblasts, responsible for matrix formation, the mast cells, involved in local response to inflammatory stimuli, resident macrophages, plasma cells, lymphocytes, granulocytes and monocytes, all engaged in immunity responses. Recently, another type of interstitial cell, found in all organs so far examined, has been added to the previous ones, the telocytes (TC). In the gut, in addition to the cells listed above, there are also the interstitial cells of Cajal (ICC), a peculiar type of cell exclusively detected in the alimentary tract with multiple functions including pace-maker activity. The possibility that TC and ICC could correspond to a unique cell type, where the former would represent an ICC variant outside the gut, was initially considered, however, further studies have clearly shown that ICC and TC are two distinct types of cells. In the gut, while the features and the roles of the ICC are established, part of the scientific community is still disputing these ‘new’ interstitial cells to which several names such as fibroblast-like cells (FLCs), interstitial Cajal-like cells or, most recently, PDGFRα+ cells have been attributed. This review will detail the main features and roles of the TC and ICC with the aim to establish their relationships and hopefully define the identity of the TC in the gut.
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Jaime-Sanchez, Paula, Iratxe Uranga-Murillo, Nacho Aguilo, Sofia C. Khouili, Maykel A. Arias, David Sancho, and Julian Pardo. "Cell death induced by cytotoxic CD8+T cells is immunogenic and primes caspase-3–dependent spread immunity against endogenous tumor antigens." Journal for ImmunoTherapy of Cancer 8, no. 1 (April 2020): e000528. http://dx.doi.org/10.1136/jitc-2020-000528.
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BackgroundElimination of cancer cells by some stimuli like chemotherapy and radiotherapy activates anticancer immunity after the generation of damage‐associated molecular patterns, a process recently named immunogenic cell death (ICD). Despite the recent advances in cancer immunotherapy, very little is known about the immunological consequences of cell death activated by cytotoxic CD8+T (Tc) cells on cancer cells, that is, if Tc cells induce ICD on cancer cells and the molecular mechanisms involved.MethodsICD induced by Tc cells on EL4 cells was analyzed in tumor by vaccinating mice with EL4 cells killedin vitroorin vivoby Ag-specific Tc cells. EL4 cells and mutants thereof overexpressing Bcl-XLor a dominant negative mutant of caspase-3 and wild-type mice, as well as mice depleted of Tc cells and mice deficient in perforin, TLR4 and BATF3 were used.Ex vivocytotoxicity of spleen cells from immunized mice was analyzed by flow cytometry. Expression of ICD signals (calreticulin, HMGB1 and interleukin (IL)-1β) was analyzed by flow cytometry and ELISA.ResultsMice immunized with EL4.gp33 cells killed in vitro or in vivo by gp33-specific Tc cells were protected from parental EL4 tumor development. This result was confirmed in vivo by using ovalbumin (OVA) as another surrogate antigen. Perforin and TLR4 and BATF3-dependent type 1 conventional dendritic cells (cDC1s) were required for protection against tumor development, indicating cross-priming of Tc cells against endogenous EL4 tumor antigens. Tc cells induced ICD signals in EL4 cells. Notably, ICD of EL4 cells was dependent on caspase-3 activity, with reduced antitumor immunity generated by caspase-3–deficient EL4 cells. In contrast, overexpression of Bcl-XLin EL4 cells had no effect on induction of Tc cell antitumor response and protection.ConclusionsElimination of tumor cells by Ag-specific Tc cells is immunogenic and protects against tumor development by generating new Tc cells against EL4 endogenous antigens. This finding helps to explain the enhanced efficacy of T cell-dependent immunotherapy and provide a molecular basis to explain the epitope spread phenomenon observed during vaccination and chimeric antigen receptor (CAR)-T cell therapy. In addition, they suggest that caspase-3 activity in the tumor may be used as a biomarker to predict cancer recurrence during T cell-dependent immunotherapies.
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Hasan, Aisha, Annamalai Selvakumar, Bo Dupont, Michel Sadelain, Isabelle Riviere, and Richard J. O’Reilly. "IL-15 Augments in-Vitro Expansion and Functional Activity of Antigen-Specific Effector Memory T-Cells (TEM) While Co-Expression of IL-15 and IL-15 Rα on Antigen Presenting Cells Also Promotes Expansion of Central Memory T-Cells (TCM)." Blood 112, no. 11 (November 16, 2008): 3541. http://dx.doi.org/10.1182/blood.v112.11.3541.3541.
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Abstract Adoptive immunotherapy with in-vitro expanded antigen-specific T cells (TC) is often hampered by the extended culture times required to generate sufficient numbers of antigen- specific TC and their limited persistence in-vivo. IL-2 predominantly supports the generation of short-lived effector memory (TEM) and effector (TE) CD8+ TC without expanding the CD62L+ and CCR7+ central memory T cells (TCM), which may persist for long periods in vivo. We examined the potential of IL-15 to foster growth of CMVpp65- specific TC as well as expansion of both TEM and TCM CD8+ TC. Accordingly, TC from 3 seropositive donors bearing HLA A0201 were sensitized in-vitro using NIH 3T3 based artificial antigen presenting cells transduced to express B7.1, ICAM-1, LFA-3, β2-M and HLA A0201 heavy chain as well as CMVpp65 (A2-AAPC) in presence of exogenous IL-2, IL-15 or IL-15 plus IL-2. To assess if trans-presentation of IL-15 by the receptor alpha on AAPCs leads to enhancement of IL-15 activity, we sequentially transduced A2-AAPCs with the IL-15 and IL-15 receptor alpha cDNA (A2- AAPC IL-15/IL-15Rα). TC were then cultured using A2-AAPC with either IL-2 (20U/ml) IL-15 (10ng/ml) IL-2 plus IL-15 or with A2-AAPC IL-15/IL-15Rα or A2-AAPC IL-15Rα + IL-2 (20U/ml). TC sensitized using either A2-AAPC IL-15/IL-15Rα or A2-AAPC + IL-15 demonstrated ~ 1500–2000 fold expansion of CMVpp65 A2-NLV tetramer (+) CD8+ TC, compared to a 300–600 fold expansion using A2-AAPC + IL-2 after 21–28 days in culture. Cultures containing IL-15 generated 25–70 ×106 A2-NLV tetramer (+) CD8+ TC in comparison to 10–18 ×106 in cultures with IL-2. At 7–10 days, ~20% of the tetramer (+) TC demonstrated a TCM phenotype (CD62L+ and CCR7+) in all cultures, with 75% TEM and 5% TE. By 21- 28 days, no TCM were detected among tetramer (+) TC in cultures containing exogenous IL-2, IL-15 or IL-2 plus IL-15. However, TC sensitized with A2-AAPC IL-15/IL-15Rα still contained 10–12% (~7×106) tetramer (+) CD8+ TCM which further increased through day 35. In functional assays, TC sensitized in the presence of IL-15 or AAPCs expressing IL-15 and IL-15Rα elicited superior CMV-specific responses with 7–20% CD8+ TC demonstrating CMVpp65 epitope specific interferon gamma production compared to 2–3% in cultures with IL-2. Cytotoxic activity against CMV pp65 peptide loaded autologous EBV transformed B-cell lines (E:T= 10:1) was higher for TC sensitized with IL-15 (80%) compared to cultures with IL-2 (60%). The cytotoxic activity against HLA mismatched targets or K562 was <5% in all cultures. In conclusion, our data demonstrate higher yields and augmented function in antigen specific T cells cultured with IL-15. TC sensitized with A2-AAPC-CMVpp65 together with IL-15 +/− IL-2 only supported sustained expansion of TEM and TE. In contrast, TC sensitized with A2-AAPC IL-15/IL-15Rα also supported sustained expansion of TCM.
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Mueller, Antonia MS, Mareike Florek, and Judith Shizuru. "Engraftment Failure After Non-Myeloablative Hematopoietic Stem Cell Transplantation: Donor CD4 Cells Interfere with Residual Host Cells and Can Trigger Rejection of the Graft." Blood 114, no. 22 (November 20, 2009): 3528. http://dx.doi.org/10.1182/blood.v114.22.3528.3528.
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Abstract Abstract 3528 Poster Board III-465 In the last decade the field of allogeneic hematopoietic cell transplantation has made significant strides in the reduction of morbidity and mortality using non-myeloablative conditioning. However, with reduced conditioning intensity the risk of allograft rejection has increased due to the persistence of host cells that mediate host-vs-graft alloreactivity or that occupy niches in the host hematopoietic microenvironment. Here we studied hematopoietic reconstitution and chimerism after non-myeloablative total body irradiation (TBI: 4 Gy [sublethal]; 7 Gy [lethal]) or total lymphoid irradiation (TLI: 17×240 cGy) in a MHC-matched mouse model with known high resistance to engraftment. BALB.K (H-2K) hosts received FACS-purified hematopoietic stem cells (HSC: cKIT+Thy1.1loSca1+Lin-) alone or supplemented with T cells (TC) from AKR/J (H-2K) donors. Peripheral blood (PB), bone marrow (BM), and spleen (SP) were analyzed 2 and 4 weeks (w) post transplant (pTX), and PB chimerism was followed beyond day (d) 100. Mice conditioned with lethal TBI survived, when grafts contained HSC only, and became mixed chimeric for TC, but converted to primarily donor type for B cells and the myeloid lineage. When grafts were supplemented with TC, chimerism converted to full donor type, but hosts developed fulminant acute GVHD. In contrast, sublethal TBI resulted in only mild symptoms of GVHD and mice recovered rapidly. Recipients of HSC alone or HSC+CD8 TC became mixed chimeras in all lineages, even long-term (>100d). However, mice given grafts of either HSC with total TC, or HSC+CD4 TC remained host type for T and B cells, and achieved only low levels of donor engraftment in the myeloid lineage. These findings suggested that the addition of peripheral donor CD4 TC resulted in powerful lymphoid suppression as well as retardation of myeloid engraftment. Indeed at 2w pTX recipients of HSC+CD4 TC displayed severe lymphopenia (<5% of live cells [vs >40% in recipients of HSC alone]) and B cell reconstitution was the most severely affected (6% vs 75% of lymphocytes). Graft suppression was also evidenced by absolute cytopenia as mice given HSC+CD4 TC vs HSC alone, had reduced cellularity in BM (median 3.8×10∧6 [n=7] vs 13.3 ×10∧6 cells [n=4]; p=0.0003) and spleen (median 4.4×10∧6 vs 14×10∧6 cells; p=0.03). Suppression of hematopoiesis by CD4 TC grafts was accompanied by an expansion of donor and host CD4 TC, each comprising ∼6% of BM cells as compared with <1% in groups that received pure HSC or were only irradiated sublethally. PMA-stimulated donor, but not host CD4 TC secreted high levels of IFNγ (30-50%) in the BM. This IFNγ expression far exceeded the levels of IFNγ measured in CD4 TC of the spleen from treated (HSC+CD4) mice as well as in CD4 TC of BM or spleen from transplanted, irradiated or unmanipulated control mice (3-15%). The proportion of NK cells in the BM was also substantially increased in recipients of HSC+CD4 TC vs recipients of HSC only or sublethal TBI controls (median 14% vs <1%), as were the absolute NK cell numbers (6.3×10∧5 vs <0.4 ×10∧5 cells). NK cells were derived from both donor and host in recipients of HSC, but solely of host type in recipients of HSC+CD4 TC. Findings similar to those observed in the 4 Gy TBI-treated mice given donor CD4 TC with their graft were noted when mice underwent non-myeloablative TLI for conditioning: while recipients of HSC alone were mixed chimeras in their lymphoid and myeloid lineages [n=4], mice that received HSC supplemented with splenocytes or total TC failed to engraft with T and B cells, had marginal myeloid engraftment [n=12], but had increased proportions of host NK cells in the PB. In conclusion: Our data show in the setting of non-myeloablative conditioning transplantation of purified HSC alone or with CD8 TC results in superior immune reconstitution and donor chimerism as compared to grafts of HSC+CD4 or HSC+total TC. These results are of broad significance as it is generally believed that TC augment donor HSC engraftment. The way in which CD4 TC retard engraftment appears to be by activation of inflammation within the BM and expansion of host lymphoid populations that may mediate resistance. IFNγ, which is a known regulator of innate and acquired immune responses, may be central in activating host CD4 TC and enhancing NK cell mediated rejection of the graft and/or suppression of donor hematopoiesis. Disclosures: No relevant conflicts of interest to declare.
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Vackova, Julie, Ingrid Polakova, Shweta Dilip Johari, and Michal Smahel. "CD80 Expression on Tumor Cells Alters Tumor Microenvironment and Efficacy of Cancer Immunotherapy by CTLA-4 Blockade." Cancers 13, no. 8 (April 16, 2021): 1935. http://dx.doi.org/10.3390/cancers13081935.
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Cluster of differentiation (CD) 80 is mainly expressed in immune cells but can also be found in several types of cancer cells. This molecule may either activate or inhibit immune reactions. Here, we determined the immunosuppressive role of CD80 in the tumor microenvironment by CRISPR/Cas9-mediated deactivation of the corresponding gene in the mouse oncogenic TC-1 cell line. The tumor cells with deactivated CD80 (TC-1/dCD80-1) were more immunogenic than parental cells and induced tumors that gained sensitivity to cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade, as compared with the TC-1 cells. In vivo depletion experiments showed that the deactivation of CD80 switched the pro-tumorigenic effect of macrophages observed in TC-1-induced tumors into an anti-tumorigenic effect in TC-1/dCD80-1 tumors and induced the pro-tumorigenic activity of CD4+ cells. Moreover, the frequency of lymphoid and myeloid cells and the CTLA-4 expression by T helper (Th)17 cells were increased in TC-1/dCD80-1- compared with that in the TC-1-induced tumors. CTLA-4 blockade downregulated the frequencies of most immune cell types and upregulated the frequency of M2 macrophages in the TC-1 tumors, while it increased the frequency of lymphoid cells in TC-1/dCD80-1-induced tumors. Furthermore, the anti-CTLA-4 therapy enhanced the frequency of CD8+ T cells as well as CD4+ T cells, especially for a Th1 subset. Regulatory T cells (Treg) formed the most abundant CD4+ T cell subset in untreated tumors. The anti-CTLA-4 treatment downregulated the frequency of Treg cells with limited immunosuppressive potential in the TC-1 tumors, whereas it enriched this type of Treg cells and decreased the Treg cells with high immunosuppressive potential in TC-1/dCD80-1-induced tumors. The immunosuppressive role of tumor-cell-expressed CD80 should be considered in research into biomarkers for the prediction of cancer patients’ sensitivity to immune checkpoint inhibitors and for the development of a tumor-cell-specific CD80 blockade.
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Kos, F. J., and A. Müllbacher. "IL-2-independent activity of IL-7 in the generation of secondary antigen-specific cytotoxic T cell responses in vitro." Journal of Immunology 150, no. 2 (January 15, 1993): 387–93. http://dx.doi.org/10.4049/jimmunol.150.2.387.
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Abstract Purified CD8+ splenocytes from influenza virus strain A/WSN/33 (H1N1)-immune BALB/c (H-2d) mice responded to a synthetic peptide, synthetic influenza nucleoprotein peptide 147-158 R-, with a sequence (147-158 R156-) derived from influenza A virus nucleoprotein with high affinity for Kd class I MHC molecules, with the generation of effector cytotoxic T (Tc) cells specific for influenza A virus-infected target cells in vitro. The process of the conversion of synthetic influenza nucleoprotein peptide 147-158R- -Kd-responding memory Tc into effector Tc cells requires Ag in the form of peptide associated with Kd class I MHC molecules and the presence of endogenously produced IL-2 by CD8+ T cells. Under blockade of utilization of endogenous IL-2 by mAb to IL-2 or IL-2R, no effector Tc cells were generated, but the addition of IL-7 restored the process of the conversion of CD8+ memory into effector Tc cells and significantly enhanced the specific cytolytic activity of Tc cells above those of controls. IL-7-reactive cells were found in both IL-2Rhigh- and IL-2Rlow-expressing responder CD8+ T cells. We conclude that the requirement for endogenous IL-2 in Ag-induced conversion of CD8+ memory Tc cells into effector Tc cells can be replaced by exogenous IL-7. This demonstrates that IL-7 is a potent regulatory cytokine with similar activity to IL-2 and may act independently of IL-2 in the Ag-specific activation of memory CD8+ Tc cells.
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Jimenez, David Gomez, Aastha Sobti, David Askmyr, Christina Sakellariou, Sofia Carreira Santos, Sabine Swoboda, Ola Forslund, Lennart Greiff, and Malin Lindstedt. "Tonsillar Cancer with High CD8+ T-Cell Infiltration Features Increased Levels of Dendritic Cells and Transcriptional Regulation Associated with an Inflamed Tumor Microenvironment." Cancers 13, no. 21 (October 25, 2021): 5341. http://dx.doi.org/10.3390/cancers13215341.
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Human papillomavirus (HPV) is the main causal agent of tonsillar cancer (TC) and HPV+ TC has a favorable prognosis compared to HPV− disease. In this study, we examined aspects of the tumor microenvironment of TC, focusing on T-cells, dendritic cells (DC), and macrophages. Fresh biopsies of TC and the contralateral healthy tonsil (HT) were obtained from 20 patients, analyzed by multiparameter flow cytometry, and assessed against a detailed HPV-status. Additionally, RNA-sequencing data from 38 TC samples available in the public database, The Cancer Genome Atlas (TCGA), were explored, focusing on the same leukocyte populations. HPV+ TC featured increased levels of CD8+ T-cells and antigen-presenting cells (cf. HPV− TC and HT, respectively). In HPV+ TC, CD8+ T-cell frequencies correlated to DC levels independently of tumor stage, HPV 16 copy number, and E7 oncogene expression as well as frequencies of other leukocytes. Similarly, RNA sequencing data were explored by dividing the HPV+ TCs according to predefined CD8+ T-cell scores in silico. Higher levels of genes expressed by antigen-presenting cells and effector T-cells, such as immune checkpoints and cytokines, were detected in the CD8HIGH HPV+ TC samples (cf. CD8LOW HPV+ TC). In conclusion, CD8HIGH HPV+ TC displays a unique inflammatory profile associated with increased effector T-cell functions and the presence of antigen-presenting cells in the tumor microenvironment. Further studies are warranted to assess if this information can be used on an individual basis to aid in prognosis and treatment decisions.
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Schwarz, Sandra, Johanna Schmitz, Markus W. Löffler, Michael Ghosh, Hans-Georg Rammensee, Evgenia Olshvang, Marvin Markel, et al. "T cells of colorectal cancer patients’ stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells." Journal for ImmunoTherapy of Cancer 10, no. 12 (December 2022): e005651. http://dx.doi.org/10.1136/jitc-2022-005651.
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BackgroundPatients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc).MethodsColorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients’ Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay.Results100 tumor-specific candidate peptides—97 cryptic peptides and 3 classically mutated neoantigens—were selected. The neoantigens originated from single nucleotide substitutions in the genesIQGAP1, CTNNB1,andTRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment.ConclusionThese results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.
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Young, Ryan M., Avital Polsky, and Yosef Refaeli. "TC-PTP is required for the maintenance of MYC-driven B-cell lymphomas." Blood 114, no. 24 (December 3, 2009): 5016–23. http://dx.doi.org/10.1182/blood-2008-12-196709.
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Abstract We sought to determine the contributions of protein tyrosine phosphatases (PTPs) to the pathogenesis of B-cell lymphomas. We found that T-cell PTP (TC-PTP) was overexpressed in transformed B cells. We hypothesized that TC-PTP may be a tumor-promoting gene that is regulated by MYC overexpression in B cells. Knockdown of TC-PTP in murine tumors resulted in decreased cell viability in vitro because of an arrest in the G1 phase of the cell cycle. Furthermore, cells with reduced TC-PTP expression were unable to either engraft or expand in vivo. Taken together, these data indicate that TC-PTP is required for B-cell tumor maintenance. Our data also suggested a correlation between TC-PTP expression and MYC overexpression. To investigate this further, we used malignant murine B cells that contain a doxycycline-repressible MYC transgene. We found that repression of MYC overexpression with doxycycline reduced TC-PTP expression. Moreover, enforced expression of TC-PTP showed partial rescue of the expansion of tumor cells after suppression of MYC overexpression. These results suggest that MYC overexpression induces TC-PTP overexpression, which in turn promotes tumor proliferation, implicating TC-PTP as an important effector of the MYC-driven proliferation program in B-cell lymphomas. Thus, TC-PTP may be a suitable molecular target for the treatment of B-cell lymphomas.
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Mueller, Antonia MS, Holbrook E. Kohrt, Sumana Shashidhar, Mareike Florek, Natascha J. Kuepper, Janice (Wes) M. Brown, and Judith A. Shizuru. "Graft T Cells Retard Stem Cell Derived Lymphoid Organ Reconstitution and Immune Function After Allogeneic Transplantation,." Blood 118, no. 21 (November 18, 2011): 4030. http://dx.doi.org/10.1182/blood.v118.21.4030.4030.
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Abstract Abstract 4030 Impaired immune function is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Adoptively transferred donor T cells (TC) mediate graft-versus-host disease (GVHD), and make the use of pharmacological immunosuppression necessary, yet are believed to protect against infections post-HCT (pTX). In an MHC-matched, minor antigen mismatched mouse model, we studied the impact of allogeneic doTC on GVH-related damage of lymphoid organs, their recovery and immune function pTX. Lethally irradiated BALB.B mice received purified hematopoietic stem cells (HSC; cKit+Sca1+Thy1.1loLin−) +/− TC from C57BL/6 (B6) donors. Recipient lymphoid organs, marrow and livers were analyzed at multiple time points. Mice given HSC+TC promptly became full donor chimeras in all lineages and developed moderate GVHD. HSC recipients retained some residual host cells, and had no signs of GVHD. By tetramer analysis and ELISPOT assay, we showed that in pure HSC recipients early pTX residual host TC protected against murine cytomegalovirus infection. Once a nascent donor HSC-derived TC pool was established, these TC reacted robustly against the virus. In contrast, transferred mature allogeneic TC proliferated and expanded in HSC+TC recipients, but did not respond or protect against the virus. To better understand why MHC-matched mature donor TC did not react against this pathogen, even when derived from pre-immunized donors, we studied the lymphatic microenvironment, hypothesizing that only highly organized interactions between cellular and humoral immunity, antigen-presenting and innate immune cells provide full immune function. Comparing tissues of mice given pure HSC +/− TC we found that: (i) lethal radiation caused marked reduction in lymphoid organ cellularity at 2 wks pTX in all transplanted groups. While the cellularity increased rapidly in HSC recipients, severe hypocellularity persisted in HSC+TC recipients, was associated with significantly decreased organ size (lymph nodes [LN] and thymuses) and marked disruption of the histological architecture; (ii) TC phenotypes present in reconstituted mice comprised a mixture of naïve (CD62L+CD44−), central memory (CM; CD62L+CD44+) and effector memory (EM; CD62L−CD44+) cells in HSC recipients, but were largely EM and rarely naïve TC in HSC+TC recipients; (iii) at ∼3 wks pTX donor B cells (BC) dominated lymphoid tissues in HSC recipients, but were absent in HSC+ToTC recipients; (iv) NK cells regenerated promptly and steadily in HSC recipients, constituting up to 20% of all lymphoid cells in the liver at 3 wks pTX, while in HSC+TC recipients they incessantly decreased to near-complete absence; (v) in mesenteric (m) LN of HSC-, but not HSC+TC- recipients, CD4+CD8+CD3− TC were observed, derived from donor HSC. This latter population resembled immature thymic TC, suggesting that peripheral lymphoid organs participate in lymphoid reconstitution in adult recipients; (vi) at 2 wks pTX we noted only in HSC, but not HSC+ToTC recipients persisting and regenerating innate lymphoid cells (ILC). No ‘classical' lymphoid tissue inducer cells, as described in embryogenesis of lymphoid organs, but cells with features of ILC were identified. Donor HSC-derived NKp46+RORgt+CD3− NK-like cells and IL-17A-secreting residual host CD4 TC were present, especially in livers and mLN. Cells that secreted IL-17A only and IL-17A+IFNγ were identified. IL-17A was not detected in recipients of HSC+ToTC, rather their donor TC were characterized by high IFNγ levels. Our studies show that, paradoxically, pure HSC grafts have advantages over TC-replete grafts with regard to immune function pTX. We hypothesize this superiority may be due to the presence of ILC that participate in the repair of lymphoid organs, and the whole spectrum of immune cells, including TC, BC, innate and immature cells emerging in HSC recipients. An intact microenvironment appears to be critical for optimal activation of TC to respond to pathogens. Moreover, our studies imply that donor TC cannot exert their protective immune function, when rapidly expanding alloreactive TC prevent the development of other lymphoid and innate immune cell, and disrupt the lymphoid tissue architecture. Controlling the negative effects of mature donor TC remains a challenge, and requires better understanding of the complexity of functional immune reconstitution pTX. Disclosures: No relevant conflicts of interest to declare.
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Pellegrini, Maria-Simonetta Faussone, and Laurenţiu M. Popescu. "Telocytes." BioMolecular Concepts 2, no. 6 (December 1, 2011): 481–89. http://dx.doi.org/10.1515/bmc.2011.039.
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AbstractHere, we review the history, morphology, immunohistochemical phenotype, and presumptive roles of a new type of interstitial tissue cells, formerly called interstitial Cajal-like cells (ICLC) and by 2010 named ‘telocytes’ (TC). Many different techniques have been used to characterize TC and provide their unequivocal identification: (i) in vitro, cultures and isolated cells; (ii) in situ, fixed specimens examined by light and fluorescence microscopy, transmission (TEM) and scanning electron microscopy, and electron tomography. TEM allowed sure identification and characterization of the most peculiar feature of TC: the long, thin, and convoluted prolongations named ‘telopodes’. An enormous variety of antibodies have been tested, but presently none are reliable to specifically label TC. TC have a mesenchymal origin and are resident connective tissue (stromal) cells. Possible identification with ‘already identified’ stromal cell types (fibroblasts, fibrocytes, fibroblast-like cells, and mesenchymal stromal cells) is discussed. We conclude that in adulthood, most of the TC have the morphology of fibrocytes. Apparently, immunocytochemistry suggests that a variety of TC populations showing different, likely organ-specific, immunophenotypes might exist. Several roles have been hypothesized for TC: mechanical roles, intercellular signaling, guiding and nursing of immature cells during organogenesis, and being themselves a pool of precursors for many of the mesenchyme-derived cells in adulthood; however, none of these roles have been proven yet. On the basis of the available data, we propose TC may be key players in organ regeneration and repair.
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Schuetz, F., C. Sachsenweger, J. Rom, A. Schneeweiss, V. Schirrmacher, P. Beckhove, and C. Sohn. "Interactions of breast cancer cells and tumorantigen-reactive T-cells derived from bone marrow." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 13517. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.13517.
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13517 Breast cancer is an immunogenic tumor which is usually recognized by the cellular immunosystem via tumor-associated antigens (TAA) presented by antigen-presenting cells like dendritic cells. Although we were able to find tumorantigen-reactive CD8+CD45R0+ T-memory cells (TMC) in the bone marrow of 67% of primary breast cancer patients by using interferon-?-ELISPOT-analysis there seem to be a minority of non-responers. In comparison to classic tumor characteristics non-responders can be find more often in non-differentiated, hormone-receptor negative tumors. In a phase-1 trial of a cellular immunotherapy with reactivated tumorantigen-reactive autologous TMC derived from bone marrow we measured CD4+ T-cell (TC) responses in stimulation cultures ex vivo to examine whether there are other immunological answers in non-responder. TC were activated by dendritic cells pulsed with TAA from MCF-7 lysate under IL-2 co-stimulation. We were able to show that next to a classic TH1-response with high levels of IFN-a there seems to exist TH2-responses mediated by high levels of TGF-β1 and low levels of IFN-a. The relation of both cytokines was directly related to the detection of tumorantigen-reactive TC and to tumor grading. Multiplex-cytokine analysis was able to confirm these findings. Additionally there seems to be an important impact of regulatory TC in responders as well as in non-responders. In patients with tumorantigen-reactive TC a combined active and passive vaccination therapy was done. These results may play an important role in further active and passive vaccination strategies. No significant financial relationships to disclose.
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Li, Fenglei, and Kaushik Choudhuri. "TCR-enriched microvesicles provide antigen-specific help for class-switched antibody production." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 166.20. http://dx.doi.org/10.4049/jimmunol.208.supp.166.20.
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Abstract Extracellular vesicles have shown essential roles in various biological processes, mainly through mediating cell-cell communications by its surface and internal cargo. Recently it was found that activated T cells release TCR enriched microvesicles (Tc-MV) from immunological synapse, but the function of Tc-MV is still a mystery. By analyzing proteomics and miRNA profiles of Tc-MV derived from mouse OT-II CD4 T cells (OVA323–339 specific), we found Tc-MV are enriched with proteins related to T cell helper function, including TCRs, CD40L, CD28, ICOS, OX40 and LFA-1, and also contained B cell function involved miRNAs, such as miR-21a-5p, miR-28a-3p, miR-155-5p and miR-210-3p. Accordantly, by in vitro coculture, OT-II Tc-MV could promote NP-specific B cells to secret IgG when cognate antigen NP-OVA was present, similarly Tc-MV from SMARTA T cell (LCMV GP61–80 specific) could promote B cells with antigen NP-GFP-GP61–80, but switching either the MV or the antigens in these two systems dampened the antibody production, suggesting Tc-MV help B cells in an antigen specific manner. The helper function of the Tc-MV was also confirmed in vivo by serum antibody production of Rag1−/− mice transferred with Tc-MV and B cells and followed by immunization, importantly the endogenous Tc-MV were found in the serum of the WT mice post-immunization, demonstrating a fundamental role of Tc-MV in the physiological condition. In conclusion, our findings revealed a critical role of Tc-MV in promoting antigen specific antibody production of B cells, which can be exploited for vaccine design. Supported by University of Michigan Startup Funds and NIH: R01AI134999.
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Song, ZX, RK Shadduck, DJ Jr Innes, A. Waheed, and PJ Quesenberry. "Hematopoietic factor production by a cell line (TC-1) derived from adherent murine marrow cells." Blood 66, no. 2 (August 1, 1985): 273–81. http://dx.doi.org/10.1182/blood.v66.2.273.273.
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Abstract An adherent cell line, termed TC-1, has been isolated from long-term liquid culture of murine marrow cells by repeated exposure of the adherent cells to 0.1% trypsin. This is an alkaline phosphatase- positive cell line showing variable staining with acid phosphatase and alpha-naphthyl acetate esterase. On electron microscopy, the cells have moderate amounts of rough endoplasmic reticulum and variable numbers of polyribosomes. Some cells contain large clusters of laked glycogen particles. Intermediate junctions are present between some cells. Conditioned medium from this cell line produced from 384 to 638 units of CSF-1 per milliliter by radioimmunoassay and a CSF-1-dependent synergistic activity, which stimulates giant macrophage colony formation of marrow cells in soft agar. The conditioned medium also stimulates 3H-TdR incorporation by marrow cells in liquid culture and induces secondary adherent cell lines. The growth factor(s) produced by the TC-1 stromal cell line may be important in the regulation of early stages of hematopoietic differentiation. Two subclones, TC-1-C-11 and TC-1-C-3, have been isolated from passage 25 of the TC-1 cells by a penicylinder separation technique. The TC-1-C-11 is phenotypically like the parent TC-1 line and produces macrophage growth factors. The TC-1-C- 3 grows as an epithelioid monolayer with visible junctions among adjacent cells under phase contrast microscopy. This subclone produces retrovirus and is capable of providing anchorage support for hematopoietic stem cells. The TC-1 cell line and its subclones may provide models for the control of early stem cell proliferation and differentiation.
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Song, ZX, RK Shadduck, DJ Jr Innes, A. Waheed, and PJ Quesenberry. "Hematopoietic factor production by a cell line (TC-1) derived from adherent murine marrow cells." Blood 66, no. 2 (August 1, 1985): 273–81. http://dx.doi.org/10.1182/blood.v66.2.273.bloodjournal662273.
Full textAbstract:
An adherent cell line, termed TC-1, has been isolated from long-term liquid culture of murine marrow cells by repeated exposure of the adherent cells to 0.1% trypsin. This is an alkaline phosphatase- positive cell line showing variable staining with acid phosphatase and alpha-naphthyl acetate esterase. On electron microscopy, the cells have moderate amounts of rough endoplasmic reticulum and variable numbers of polyribosomes. Some cells contain large clusters of laked glycogen particles. Intermediate junctions are present between some cells. Conditioned medium from this cell line produced from 384 to 638 units of CSF-1 per milliliter by radioimmunoassay and a CSF-1-dependent synergistic activity, which stimulates giant macrophage colony formation of marrow cells in soft agar. The conditioned medium also stimulates 3H-TdR incorporation by marrow cells in liquid culture and induces secondary adherent cell lines. The growth factor(s) produced by the TC-1 stromal cell line may be important in the regulation of early stages of hematopoietic differentiation. Two subclones, TC-1-C-11 and TC-1-C-3, have been isolated from passage 25 of the TC-1 cells by a penicylinder separation technique. The TC-1-C-11 is phenotypically like the parent TC-1 line and produces macrophage growth factors. The TC-1-C- 3 grows as an epithelioid monolayer with visible junctions among adjacent cells under phase contrast microscopy. This subclone produces retrovirus and is capable of providing anchorage support for hematopoietic stem cells. The TC-1 cell line and its subclones may provide models for the control of early stem cell proliferation and differentiation.
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Durak, Hatice, Oĝuz Kilinç, Türkan Ertay, Eyüp Sabri Uçan, Aydanur Kargi, Gamze Çapa Kaya, and Banu Sis. "Tc-99m-HMPAO uptake by bronchoalveolar cells." Annals of Nuclear Medicine 17, no. 2 (April 2003): 107–13. http://dx.doi.org/10.1007/bf02988447.
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Sun, Caixia, Ping Wang, Tiantian Gao, and Jinfeng Chi. "CCL18 Knockdown Suppresses Cell Growth and Migration in Thyroid Cancer." Journal of Healthcare Engineering 2022 (January 25, 2022): 1–8. http://dx.doi.org/10.1155/2022/1548155.
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Background. Chemokine (C-C motif) ligand 18 (CCL18) is a chemokine that plays a key role in immune and inflammatory responses. In recent years, CCL18 participates in the development and progression of various cancers, but its expression and role in thyroid cancer (TC) remain unclear. Methods. RT-qPCR assay and Western blot assay were used to explore the expression level of CCL18 in TC tissues and cells. Cell proliferation was measured by MTT assay. Transwell assay was adopted to detect cell migration in TC cells. Dual luciferase reporter assay was performed to assess the relationship between CCL18 and miR-149-5p. Results. There was an uptrend of CCL18 in TC tissues and cells. Our findings indicated that CCL18 overexpression facilitated lymph node metastasis in patients with TC. CCL18 silencing was found to inhibit cell migration, proliferation, and EMT progression in TC cells. CCL18 was proved to be a target gene of miR-149-5p. Additionally, miR-149-5p weakened the effect of CCL18 in the progression of TC. Conclusion. Therefore, our results indicated that CCL18 knockdown restrained TC progression and suggested that CCL18 might be a potential therapeutic target for TC.
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Liu, Di, Rukhsanda Aziz, Md Jahidul Islam Shohag, Lingli Lu, Yuyan Wang, Ying Feng, Tingqiang Li, et al. "Assessment of Indicators in a Human Liver Cell Line HL-7702 for Tetracycline Toxicity in Farm Soil." Agronomy 12, no. 3 (March 17, 2022): 730. http://dx.doi.org/10.3390/agronomy12030730.
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Tetracycline (TC) contamination has become hot research topic, but little attention has been paid to its ecotoxicological monitoring. The primary objective of this study is to investigate the impact of TC on human normal liver cells (HL-7702) and find indicators for monitoring their ecotoxicity. The cytotoxicity of TC, at concentrations ranging from 0 to 1000 μg L−1, was assessed on HL-7702 cells. The results showed that TC significantly inhibited the cell viability at a high concentration (1000 μg L−1). The TC at exposure levels ≥ 50–100 μg L−1 significantly increased the levels of extracellular catalase (CAT), malondialdehyde (MDA), alanine transaminase (ALT), and aspartate transaminase (AST), and a significantly positive correlation between the TC concentrations and the values of the above parameters was observed. Swelling of the mitochondrial cristae (MC) and rough endoplasmic reticulum (RER) and the loss of ribosomes in HL-7702 cells, were observed at high TC levels. There was a positive correlation between soil TC concentration and ALT activities. The above results suggest that TC is cytotoxic to HL-7702 cells and that extracellular ALT activities can be used as a sensitive bioindicator for monitoring soil TC contamination. We, therefore, propose that the HL-7702 cell line can be a novel tool for early antibiotics toxicity monitoring.
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Zhong, Qiaofeng, Jianzhong Shou, Jianming Ying, Yun Ling, Yue Yu, Zhirong Shen, Yun Zhang, Ning Li, Yuankai Shi, and Aiping Zhou. "High PD-L1 expression on immune cells, but not on tumor cells, is a favorable prognostic factor in urothelial carcinoma." Future Oncology 17, no. 22 (August 2021): 2893–905. http://dx.doi.org/10.2217/fon-2021-0092.
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Aims: To explore the prognostic value of high PD-L1 expression on tumor cells (TC) and tumor-infiltrating immune cells (TIIC) in urothelial carcinoma (UC). Patients & methods: 162 UC specimens were evaluated for PD-L1 expression on TIIC and TC with the SP263 assay. High PD-L1 expression was defined as ≥25% staining. Results: High PD-L1 expression on TC in UC patients with stage T1–4 disease was associated with poor overall survival. However, high PD-L1 expression on TIIC in UC patients with stage T1–4 disease revealed favorable disease-free and overall survival; more significant differences were observed in patients with stages T2–4. Multivariate analysis revealed that high PD-L1 expression on TIIC was an independent prognostic predictor for better disease-free and overall survival. Conclusion: High PD-L1 expression on TIIC, but not on TC, is a favorable prognostic factor in UC.
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Koide, J., and E. G. Engleman. "Differences in surface phenotype and mechanism of action between alloantigen-specific CD8+ cytotoxic and suppressor T cell clones." Journal of Immunology 144, no. 1 (January 1, 1990): 32–40. http://dx.doi.org/10.4049/jimmunol.144.1.32.
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Abstract We have previously demonstrated that fresh CD8+ T cells proliferate in response to autologous, alloantigen-primed CD4+ T cells, and differentiate into Ts cells, which inhibit the response of fresh T cells to the primary allogeneic stimulator cell but not irrelevant stimulators. Although such Ts do not have discernible cytolytic activity, like classical cytotoxic T cells (Tc) they express CD3 and CD8 on their surface and function in a class I MHC-restricted manner. Our study was an attempt to compare the surface phenotype and mechanism of action of Ts and Tc clones derived from the same individual. Ts clones were generated from donor JK by repeated stimulation of CD8+ T cells with an autologous CD4+ T inducer line specific for an allogeneic lymphoblastoid cell line (LCL). These clones were noncytolytic for either the inducer line or the allogeneic stimulator LCL. Tc clones, generated by direct stimulation of JK CD8+ T cells with the same allogeneic LCL, mediated potent, alloantigen-specific cytolysis. All Tc clones were alpha, beta TCR+, CD3+, CD4-, CD8+, CD11b-, and CD28+. Ts clones were also alpha, beta TCR+, CD3+, and CD8+, but in contrast to Tc clones, Ts clones were CD11b+ and CD28-. When added to MLR both Ts and Tc clones inhibited the response of fresh JK CD4+ T cells to the original but not irrelevant allogeneic LCL. However, Ts inhibited the response of only those CD4+ T cells that shared class I)MHC determinants with the Ts donor, whereas Tc inhibited the response of CD4+ T cells from all responders, regardless of HLA type. Pretreatment of Ts clones with mAb to CD2, CD3, or CD8 blocked suppression, whereas similar pretreatment of Tc clones blocked cytotoxicity in 4-h 51Cr release assays but had no effect on Tc-mediated suppression of the MLR. These results suggest that both Ts and Tc clones can inhibit the MLR but they do so through different mechanisms. Moreover, the maintenance of distinct surface phenotypes on these long term clones suggests that Ts may be a distinct sublineage of CD8+ T cells rather than a variant of CD8+ Tc.
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Zhong, Qiaofeng, Ai-Ping Zhou, Jianzhong Shou, Jianming Ying, Yuankai Shi, Yue Yu, Yun Ling, Zhirong Shen, and Yun Zhang. "Different expression and prognostic implications of PD-L1 in tumor cells and immune cells with the SP263 monoclonal antibody in Chinese urothelial carcinoma patients." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e16040-e16040. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e16040.
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e16040 Background: PD-L1 is expressed on tumor cells (TC) and tumor immune cell (IC) infiltrates. The aim of this study was to explore the expression and the clinical implications of PD-L1 in TC and IC with the SP263 monoclonal antibody and the correlation with clinicopathological features and survival prognosis in urothelial carcinoma(UC) patients. Methods: Formalin-fixed paraffin embedded tumor specimens were collected from 172 patients with UC who received nephroureterectomy or cystoscope biopsy in our center from 2009 to 2016. Tumor samples were evaluated for PD-L1 membrane expression by immunohistochemistry (IHC) using VENTANA PD-L1 (SP263) Assay performed on the automated BenchMark ULTRA platform. PD-L1 expression status was determined by PD-L1 membranous staining at any intensity on TC or IC.PD-L1 is defined as positive if either ≥25% TC or ≥25% IC express membranous PD-L1.Data was analyzed by SPSS software. Results: Among 162 patients, TC and IC infiltrate PD-L1 positivity were detected in 16 (9.9%) and 45 cases (27.8%), respectively. The PD-L1 positivity of TC in female (19.4%) was higher than that in male (7.1%), P = 0.029, and so was in the patients with lymph node metastasis (20.7%) than those without metastatic lymph node (7.5%) , P = 0.031.While these correlation were not observed with PD-L1 positivity in IC. PD-L1 positivity both in TC and IC were not related to age, smoking history, specimen type , tumor size , histology grade, pT, pN stage (P > 0.05) .With a median follow-up of 60.9 months, patients with positive PD-L1 expression in IC have more favorable DFS (NR vs 26.3 months, P = 0.032) and OS (NR vs 71.2 months, P = 0.001) than those with negative PD-L1 expression in IC. More significant differences in DFS (NR vs 18 months, P = 0.002) and OS (NR vs 29.8months,P < 0.001) were observed in patients with T2-4 and PD-L1 positive expression. However, We did not find a statistically correlation between PD-L1 positivity in TC and prognostic outcomes. Univariate and multivariate analyses revealed that patients with PD-L1 positive expression in TC had poorer DFS (HR = 2.573; 95% CI, 1.246–5.313;P = 0.011). Whereas positive PD-L1 expression in IC independently correlated with better OS (HR = 0.303; 95% CI, 0.137–0.671;P = 0.003). Conclusions: PD-L1 positivity in IC is higher than TC in urothelial tumor. Positive PD-L1 expression in IC is associated with better OS and DFS while no correlation between PD-L1 positivity in TC and outcome was observed.
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Anether, Gabriele, Inge Tinhofer, Monika Senfter, and Richard Greil. "Tetrocarcin-A—induced ER stress mediates apoptosis in B-CLL cells via a Bcl-2—independent pathway." Blood 101, no. 11 (June 1, 2003): 4561–68. http://dx.doi.org/10.1182/blood-2002-08-2501.
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Abstract Tetrocarcin-A (TC-A), an antibiotic agent isolated from actinomycetes, has recently been described to antagonize Bcl-2 functions, thereby sensitizing tumor cells to cell death signals under control of Bcl-2. In this study, we analyzed the direct proapoptotic effect of TC-A in the B-chronic lymphocytic leukemia (B-CLL) model. We focused on the signal cascade triggered by TC-A in B-CLL cells and identified activated mitochondrial as well as endoplasmatic reticulum (ER) stress signals. The expression levels of known effector molecules mediating mitochondrial signaling, such as Bax and Bid, and the antagonistic molecule Bcl-2 did not influence sensitivity of B-CLL cells to TC-A. Furthermore, the molecular chaperone and sensor of ER stress, HSP70, though significantly up-regulated in B-CLL cells undergoing TC-A—triggered apoptosis, was ineffective to exert its anti-apoptotic function described in multiple cell death pathways. Autologous T cells of B-CLL patients were significantly less sensitive to TC-A as were also T cells from healthy donors when compared with their normal B-cell fraction. Furthermore, sensitivity of B-CLL cells to TC-A treatment in vitro was dependent neither on the expression levels of CD38—a prognostic factor for survival of B-CLL patients as well as for their response to therapy—nor on the clinical stage or pretreatment status of patients. From our data showing that TC-A induced a cell death pathway via ER stress preferentially in B cells and that it acted independently of important markers of drug sensitivity and of clinical markers, we conclude that TC-A might represent an attractive candidate drug for further evaluation in preclinical trials.
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Müllbacher, A., A. B. Hill, R. V. Blanden, W. B. Cowden, N. J. King, and R. T. Hla. "Alloreactive cytotoxic T cells recognize MHC class I antigen without peptide specificity." Journal of Immunology 147, no. 6 (September 15, 1991): 1765–72. http://dx.doi.org/10.4049/jimmunol.147.6.1765.
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Abstract In this report, experiments are described to differentiate between three potential models of class I MHC allorecognition, namely 1) recognition of peptide-free MHC, 2) peptide-MHC-specific recognition, and 3) peptide-MHC-nonspecific recognition. Using a nucleoprotein peptide (NPP) with a sequence derived from influenza virus nucleoprotein with high affinity for Kd class I MHC molecules, it is shown that target cells rapidly become lysable by Kd-NPP self-restricted cytotoxic T (Tc) cells, and retain sufficient Kd-NPP complexes for at least 72 h. Kd-specific alloreactive Tc cells at the clonal and polyclonal level do not show decreased lysis of Kd-bearing targets in the continuous long term (48 h) presence of NPP. Kd-stimulator cells modified with NPP are able to induce potent Kd-NPP-specific self-restricted Tc cells, however Kd-NPP stimulator cells do not generate Kd-NPP specific alloreactive Tc cells from CBA and B10.A (5R) mouse strains as tested by limiting dilution split clone experiments. Human cells infected with the vaccinia virus recombinant coding for the murine Kd class I MHC Ag can be lysed by murine Kd-specific alloreactive Tc cells. In addition the rate of reemergence of alloreactive and self-restricted Tc cell epitopes on virally infected target cells that had their cell-surface class I MHC Ag removed is identical. These results are consistent with model 3 namely that the majority of Tc precursor and effector cells recognize class I MHC Ag without peptide specificity.
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Ntranos, Achilles, Inna Grishkan, Dionne Robinson, Olivia Hall, Sabra Klein, Peter Calabresi, and Anne Gocke. "Dissecting fingolimod’s action: a novel immunomodulatory effect of the unphosphorylated compound on cytotoxic T cell function (P5159)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 68.7. http://dx.doi.org/10.4049/jimmunol.190.supp.68.7.
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Abstract Fingolimod (FTY), a sphingosine-1-phosphate receptor modulator, is a multiple sclerosis (MS) therapeutic that upon phosphorylation traps CCR7+ T cells in lymph nodes (LN). CCR7- effector T cells are spared, thus allowing effective infection clearance. Nonetheless, FTY-treated patients are more susceptible to viral infections, indicating a cytotoxic T (Tc) cell defect. Yet, FTY’s effect on Tc cells remains unclear. To address this question, we utilized experimental autoimmune encephalomyelitis (EAE) and a murine influenza model. FTY ameliorated EAE by sequestering T cells, but also inhibited splenic Tc cell IFNγ and Granzyme B(GrB) expression. Influenza was exacerbated and mortality was increased, as FTY inhibited Tc cell activation and lung infiltration. To elucidate its mechanism of action, murine splenocyte cultures were treated with phosphorylated and unphosphorylated FTY, as both exist in vivo. Remarkably, only the latter reduced IFNγ and GrB levels in Tc cells and inhibited their function in an LDH cytotoxicity assay. The addition of arachidonic acid rescued Tc cell function, suggesting that FTY’s effect is due to inhibition of cytosolic phospholipase A2. Herein, we demonstrate that FTY suppresses Tc cell function independently of its cell trafficking effects. This provides a novel explanation not only for the increased rate of viral infections in FTY-treated patients, but also for its efficacy in MS, as Tc cells have emerged as crucial mediators of MS pathogenesis.
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Hammerich, Linda, Judit Svensson-Arvelund, Todd Covey, and Joshua Brody. "Ibrutinib, but not acalabrutinib, inhibits anti-lymphoma T cell and NK cell function." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 136.19. http://dx.doi.org/10.4049/jimmunol.202.supp.136.19.
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Abstract Chronic Lymphocytic Leukemia (CLL) is the predominant hematologic malignancy in the U.S. BTK inhibitors (BTKi) demonstrate prolonged survival compared to standard chemotherapy, but they also show notable toxicity, including increased risk for infections. While BTKi have shown both immunosuppressive and -stimulatory effects, the overall effect on T cells (TC), including tumor-reactive TC, is not well understood. Because non-BTK TEC-family and homologous kinases have critical roles in immune cells, we tested whether differentially specific BTKi have differing immunomodulatory effects. We treated mice with ibrutinib or acalabrutinib in vivo and analyzed their immune cell repertoire. Mass cytometry revealed that acalabrutinib, but not ibrutinib, increased the number of NK cells and myeloid cells in spleens and tumors of A20-lymphoma bearing mice, while both BTKi increased TC numbers in A20 tumors. Ibrutinib inhibited the activation of CD8 TC and NK cells isolated from BTKi-treated mice and their ability to lyse tumor cells ex vivo, while acalabrutinib improved CD8 TC-mediated cytolysis. A20 tumor growth was not influenced by BTKi in vivo, but ibrutinib inhibited the ability of transferred, antigen-specific TC to induce tumor regression. To translate our findings, we tested the effect of BTKi on immunogenicity of primary human CLL/MCL cells and their ability to stimulate TC. Ibrutinib inhibited upregulation of activation markers on tumor cells to a higher extent than acalabrutinib. Similarly, only ibrutinib inhibited autologous TC activation after co-culture with tumor cells and superantigen. In conclusion, ibrutinib, but not acalabrutinib inhibits the activation and function of anti-tumor T and NK cells in vivo and in vitro.
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Qiu, Jiantai, Donia Alson, Ta-Hsien Lee, Ching-Chou Tsai, Ting-Wei Yu, Yu-Sing Chen, Ya-Fang Cheng, Chu-Chi Lin, and Scott Schuyler. "Effect of Multiple Vaccinations with Tumor Cell-Based Vaccine with Codon-Modified GM-CSF on Tumor Growth in a Mouse Model." Cancers 11, no. 3 (March 15, 2019): 368. http://dx.doi.org/10.3390/cancers11030368.
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Ectopic expression of codon-modified granulocyte-macrophage colony-stimulating factor (cGM-CSF) in TC-1 cells (TC-1/cGM-CSF), a model cell line for human papillomavirus (HPV)-infected cervical cancer cells, increased the expression level of GM-CSF and improved the efficacy of tumor cell-based vaccines in a cervical cancer mouse model. The number of vaccine doses required to induce a long-term immune response in a cervical cancer mouse model is poorly understood. Here, we investigated one, three, and five doses of the irradiated TC-1/cGM-CSF vaccine to determine which dose was effective in inducing a greater immune response and the suppression of tumors. Our findings showed that three doses of irradiated TC-1/cGM-CSF vaccine elicited slower tumor growth rates and enhanced survival rates compared with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Consistently, mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine exhibited stronger interferon gamma (IFN-γ) production in HPV E7-specific CD8+ T cells and CD4+ T cells. A higher percentage of natural killer cells and interferon-producing killer dendritic cells (IKDCs) appeared in the splenocytes of the mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine compared with those of the mice vaccinated with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Our findings demonstrate that single or multiple vaccinations, such as five doses, with irradiated TC-1/cGM-CSF vaccine suppressed the immune response, whereas three doses of irradiated TC-1/cGM-CSF vaccine elicited a greater immune response and subsequent tumor suppression.
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Nam, Ok Hyung, Jae-Hwan Kim, Sung Chul Choi, and Young Kim. "Time-Dependent Response of Human Deciduous Tooth-Derived Dental Pulp Cells Treated with TheraCal LC: Functional Analysis of Gene Interactions Compared to MTA." Journal of Clinical Medicine 9, no. 2 (February 15, 2020): 531. http://dx.doi.org/10.3390/jcm9020531.
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Pulp capping material should facilitate hard tissue regeneration on the injured pulp tissue. TheraCal LC (TC) was recently developed. Although TC has shown reliable clinical outcomes after direct pulp capping, there are still remaining concerns regarding its detrimental effect on pulp cells. Therefore, this study aimed to identify the gene expression of human deciduous tooth-derived dental pulp cells exposed to TC compared to mineral trioxide aggregate (MTA). The cells were cultured and exposed to TC and MTA for 24 and 72 h. Next, total RNA was isolated. QuantSeq 3′ mRNA-sequencing was used to examine differentially expressed genes (DEGs) in exposed to TC and MTA. Functional analysis of DEGs was performed using bioinformatics analysis. In gene ontology (GO) functional enrichment analysis, cells in TC for 24 h presented significantly enriched immune response (p < 0.001) and inflammatory response (p < 0.01) compared to MTA. TC showed enriched positive regulation of cell migration at 72 h (p < 0.001). In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, neuroactive ligand–receptor interaction (p = 1.19 × 10−7) and calcium signaling pathway (p = 2.96 × 10−5) were confirmed in the shared DEGs in TC. In conclusion, DEGs in TC may be involved in pathways associated with osteoclastogenesis and osteoclastic differentiation.
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Zhang, Jizong, Yan Zhong, Yiming Sang, and Guanghui Ren. "miRNA-144-5p/ITGA3 Suppressed the Tumor-Promoting Behaviors of Thyroid Cancer Cells by Downregulating ITGA3." Computational and Mathematical Methods in Medicine 2021 (December 13, 2021): 1–9. http://dx.doi.org/10.1155/2021/9181941.
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Objective. To ascertain the mechanism of miRNA-144-5p and ITGA3 in thyroid cancer (TC). Methods. From The Cancer Genome Atlas (TCGA), RNA expression profiles were obtained for the expression analysis of miRNAs and mRNAs in TC. qRT-PCR and western blot were utilized to measure the expression of miRNA-144-5p and ITGA3 at RNA and protein levels, respectively. The association between miRNA-144-5p and ITGA3 was validated by the dual-luciferase assay. CCK-8, scratch healing, transwell, and flow cytometry assays were employed to evaluate tumor-related cell behaviors. Results. Low-expressed miRNA-144-5p and high-expressed ITGA3 were found in TC cells relative to normal cells. miRNA-144-5p expression was positively associated with suppressive effects on proliferative, invasive, and migratory ability of TC cells while negatively associated with cell apoptosis. miRNA-144-5p inhibited ITGA3 expression in TC, and its overexpression remarkably reversed the tumor-promoting effects of overexpressed ITGA3 on the biological functions of TC. Conclusion. It is verified in our study that cell growth of TC is inhibited by the miRNA-144-5p/ITGA3 axis, which represents an underlying target for TC. This research proposed a new insight into the strategy of TC treatment.
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Bourdeau, Annie, Nadia Dubé, Krista M. Heinonen, Jean-François Théberge, Karen M. Doody, and Michel L. Tremblay. "TC-PTP–deficient bone marrow stromal cells fail to support normal B lymphopoiesis due to abnormal secretion of interferon-γ." Blood 109, no. 10 (January 18, 2007): 4220–28. http://dx.doi.org/10.1182/blood-2006-08-044370.
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Abstract The T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of the Jak/Stat cytokine signaling pathway. Our study shows that the absence of TC-PTP leads to an early bone marrow B-cell deficiency characterized by hindered transition from the pre-B cell to immature B-cell stage. This phenotype is intrinsic to the B cells but most importantly due to bone marrow stroma abnormalities. We found that bone marrow stromal cells from TC-PTP−/− mice have the unique property of secreting 232-890 pg/mL IFN-γ. These high levels of IFN-γ result in 2-fold reduction in mitotic index on IL-7 stimulation of TC-PTP−/− pre-B cells and lower responsiveness of IL-7 receptor downstream Jak/Stat signaling molecules. Moreover, we noted constitutive phosphorylation of Stat1 in those pre-B cells and demonstrated that this was due to soluble IFN-γ secreted by TC-PTP−/− bone marrow stromal cells. Interestingly, culturing murine early pre-B leukemic cells within a TC-PTP–deficient bone marrow stroma environment leads to a 40% increase in apoptosis in these malignant cells. Our results unraveled a new role for TC-PTP in normal B lymphopoiesis and suggest that modulation of bone marrow microenvironment is a potential therapeutic approach for selected B-cell leukemia.
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Specht, Christoph, Hans-Gerd Pauels, Christian Becker, and Eckehart Kölsch. "Differential Activation of CD8+Tumor-Specific Tc1 and Tc2 Cells by an IL-10-Producing Murine Plasmacytoma." Developmental Immunology 6, no. 3-4 (1998): 331–42. http://dx.doi.org/10.1155/1998/93545.
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The involvement of counteractiveCD8+T-cell subsets during tumor-specific immune responses was analyzed in a syngeneic murine plasmacytoma model.CD8+Tc cells against the immunogenic IL-10-producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced by immunization of BALB/c mice with X-irradiated ADJ-PC-5 tumor cellsin vivoandin vitro. However, the failure of recipient mice to mount a protective Tc response against the tumor during early stages of a real or simulated tumor growth is not due to immunological ignorance, but depends on the induction of tumor-specific tolerance, involving a population of tumorinducedCD8+T cells that are able to inhibit the generation of tumor-specific Tc cells in a primary ADJ-PC-5-specific MLTC, using IFN-γas a suppressive factor. Whereas most longterm cultivated CD8+ADJ-PC-5-specific Tc lines produce type-1 cytokines on stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum. Furthermore, the primaryin vitroTc response against ADJ-PC-5 cells shows characteristics of a Tc2 response. The Tc response is strictly depending on tumor-derived IL-10.CD8+Tc cells that are induced in a primary MLTC do not produce IFN-γ, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-γor IL-12. In contrast, ADJ-PC- 5-specificCD8+Tc cells from immunized mice are IFN-γproducing Tc1 cells. Since the primaryin vitroTc response against the tumor is suppressed even by the smallest numbers of irradiated ADJ-PC-5-specific Tc1 cells via IFN-γthese Tc1 cells behave similar to the suppressiveCD8+T cells that are induced during early stages of ADJ-PC-5 tumorigenesis.
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Chen, Cheng, Lu Qin, and Mei-Fang Xiao. "Long Noncoding RNA LOC554202 Predicts a Poor Prognosis and Correlates with Immune Infiltration in Thyroid Cancer." Computational and Mathematical Methods in Medicine 2022 (February 28, 2022): 1–12. http://dx.doi.org/10.1155/2022/3585626.
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Thyroid cancer (TC) is one of the widely diagnosed carcinomas in women before the age of 30. Nevertheless, there is currently a lack of specific biomarkers for predicting the prognosis of TC. Long noncoding RNAs (lncRNAs) were important regulators in human cancer progression as previously described. Unfortunately, there is little known on these lncRNAs’ functions and molecular mechanisms in TC. In our literature, we found that LOC554202 (MIR31HG) was upregulated in TC samples and correlated with clinicopathological features, including M stage, N stage, and lymph nodes examined status in TC. In addition, we found that LOC554202 overexpression was evidently correlated with high immune infiltrate levels of CD8+ T cells, macrophage, neutrophil, myeloid dendritic cells, and B cells in TC. Knockdown of LOC554202 impeded TC cell proliferation and cycle progression. We found that LOC554202 had an association with metabolic pathways, vesicle-mediated transport, tricarboxylic acid cycle, Hedgehog signaling pathway, and Hippo signaling pathway in TC. Reducing LOC554202 hindered TC cell proliferation and cycle progression. Finally, we found that LOC554202 participated in modulating the expression of the regulators of Hippo signaling and TCA pathway, such as CCND2, CCND3, SDHC, SDHD, SUCLA2, and SUCLG1. We thought that this study would largely enhance our understanding of LOC554202’s functional roles in human TC progression and immune response.
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Hasan, Aisha N., Rosanna J. Ricafort, Annamalai Selvakumar, Ekaterina Doubrovina, Isabelle Riviere, Michel Sadelain, and Richard J. O'Reilly. "Engineered Antigen- Presenting Cells Expressing Human HLA Class-II DR Alleles Support Efficient Enrichment and Expansion of Antigen Specific Th1 CD4[+] T-Cells Which Are Functionally Augmented by IL-15Rα/IL-15 Complexes." Blood 120, no. 21 (November 16, 2012): 1905. http://dx.doi.org/10.1182/blood.v120.21.1905.1905.
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Abstract Abstract 1905 Previous studies have affirmed the therapeutic efficacy of adoptively transferred antigen specific CD8+ and CD4+ T-cells (TC) against viral infections and tumors. A major challenge in optimizing this approach is to develop strategies to permit generation of CD4+ and long lived CD8+ TC of defined antigen specificity. We previously described a panel of NIH 3T3 based artifical antigen presenting cells (AAPC) for the immediate generation of HLA class-I restricted CMVpp65 specific CD8+ TC. We now describe a panel of NIH 3T3 based AAPC, each transduced to express a shared HLA DRA 0101 alpha chain and one of the following β chains of the human HLA class-II alleles DRB1 0301, 0701, 1501, 0401 and 1101. At least one of these alleles is inherited by 61% and 59% of caucasians and blacks respectively. These AAPCs were also transduced to co-express the human TC costimulatory molecules B7.1, ICAM-1 and LFA-3. Sensitization of TC from seropositive donors in the presence of IL-2 with AAPCs sharing one of these alleles, either loaded with a CMVpp65 peptide pool or transduced to express the CMV pp65 protein, resulted in 33–71 fold expansion of CMVpp65 specific CD4+ TC that exhibited a Th1 cytokine profile, producing TNF-α and IFNγ in response to the same CMVpp65 epitopes. These TC were also cytotoxic against peptide loaded HLA class-II sharing targets. Epitope mapping demonstrated that the HLA DRB1 0301 restricted TC responded to a CMVpp65 epitope known to be presented by this allele QEFFWDANDIY (aa 509–527) and to an unreported epitope DVEEDLTMTRN (aa 245–263). The DRB1 0701 restricted CD4+ TC responded to 4 different epitopes. Two of these also included nonamer peptide sequences previously reported to be presented by HLA class-I alleles; Q IFLEV QAIRE and PQYSEH PTFTS presented by HLA B44, and a third AGILARNLVPM, contained an epitope presented by HLA B0801. The fourth epitope, KYQEFFWDANDIY is known to be also presented by HLA DRB1 0301. The DRB1 1501 restricted CD4+ TC were also responsive to a known class-II epitope LLQTGIHVRVS (aa 37–55) as well as a new epitope LVSQYTPDSTP (aa 53–71). CD4+ TC from 3 donors also responded to CMVpp65 when sensitized with autologous DCs loaded with CMVpp65 peptide pool, and each recognized the same epitopes as TC sensitized with the class-II AAPCs. Supplementation of TC cultures with soluble IL-15Rα/ IL-15 complexes markedly augmented the proportion of IFNg+ CD4+ TC, while increasing concentrations of IL-2 resulted in generation of Th2 type CD4+ TC generating IL-4, IL-5 and IL-2 in response to re-stimulation with CMVpp65 peptides. This system can therefore be harnessed by cytokine modulation to selectively generate CD4+ TC with a Th1, or Th2 cytokine profile. The fact that the class-II AAPC transduced to express the full sequence of CMVpp65 are able to process and present antigenic epitopes on the surface of the expressed HLA class-II allele in the absence of the human invariant chain and HLA-DM suggests that the mouse 3T3 cells contain sufficiently homologous proteins to permit the transfer of processed peptides to human Class-II alleles for presentation. Alternatively, invariant chain independent pathways could permit delivery of certain immunogenic epitopes to the expressed class II HLA alleles. The repertoire of epitopes presented by the Class-II AAPCs with or without the invariant chain is currently under study. Our results suggest that the panel of AAPCs expressing these HLA DRB1 alleles provides a novel and rapid approach for the generation of Th1 CD4+ virus-specific TC of desired HLA class-II restriction for adoptive therapy of CMV disease to foster lasting immunity with co-infused CMVpp65-specific CD8+ TC. Class-II AAPCs used with different concentrations or types of cytokines may also be useful to generate other functional subsets of CD4+ TC to promote tolerance or enhance tumor- specific immunity. Disclosures: No relevant conflicts of interest to declare.
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Schonhoff, Christopher M., Ai Yamazaki, Simon Hohenester, Cynthia R. L. Webster, Bernard Bouscarel, and M. Sawkat Anwer. "PKCε-dependent and -independent effects of taurolithocholate on PI3K/PKB pathway and taurocholate uptake in HuH-NTCP cell line." American Journal of Physiology-Gastrointestinal and Liver Physiology 297, no. 6 (December 2009): G1259—G1267. http://dx.doi.org/10.1152/ajpgi.00177.2009.
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The cholestatic bile acid taurolithocholate (TLC) inhibits biliary secretion of organic anions and hepatic uptake of taurocholate (TC). TLC has been suggested to induce retrieval of Mrp2 from the canalicular membrane via the phosphoinositide-3-kinase (PI3K)/PKB-dependent activation of novel protein kinase Cε (nPKCε) in rat hepatocytes. The aim of the present study was to determine whether TLC-induced inhibition of TC uptake may also involve PI3K-dependent activation of PKCε in HuH7 cells stably transfected with human Na+-dependent TC-cotransporting polypeptide (NTCP) (HuH-NTCP cells). To avoid direct competition for uptake, cells were pretreated with TLC, washed, and then incubated with 3H-TC to determine TC uptake. TLC produced time- and dose-dependent inhibition of TC uptake. TLC inhibited TC uptake competitively without affecting NTCP membrane translocation. A PI3K inhibitor failed to reverse TLC-induced TC uptake inhibition and TLC-inhibited PKB phosphorylation. TLC did activate nPKCε as evidenced by increased membrane translocation and nPKCε-Ser729 phosphorylation. Overexpression of dominant negative-nPKCε reversed TLC-induced inhibition of PKB phosphorylation but not of TC uptake. Finally, cAMP prevented TLC-induced inhibition of TC uptake via the PI3K pathway, and the prevention is due to the sum of cAMP-induced stimulation and TLC-induced inhibition of TC uptake. Taken together, these results suggest that TLC-induced inhibition of PKB, but not of TC uptake, is mediated via nPKCε. Activation of nPKCε and inhibition of TC uptake by TLC are not mediated via the PI3K/PKB pathway.
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Brugnatelli, Silvia, Ilaria Turin, Mariangela Manzoni, Marcello Maestri, Enrica Montini, Laura Caliogna, Marco Paulli, et al. "Preclinical study of adoptive immunotherapy with natural killer cells in metastatic colorectal cancer." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e13103-e13103. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e13103.
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e13103 Background: Several immune-based approaches for treatment of metastatic colorectal cancer (mCRC) are currently under investigation. Aims of our study were to grow and characterize tumor cells (TC) obtained from mCRC patients, and to investigate their susceptibility to autologous natural killer (NK) cell recognition and killing in vitro. Methods: Tumor samples and peripheral blood mononuclear cells from 10 mCRC patients undergoing surgery were collected. Establishment of TC lines that are representative of the original tumor and could be expanded in vitro for a significant number of passage was performed according with our previously described method (Turin I et al, Cytotherapy 2007). CD56+CD3- NK cells were incubated overnight with medium alone or activated with IL-2 (100U/ml) or IL-15 (20ng/ml) and tested against 51Cr-labeled primary TC in NK assay or in ADCC assay after pre-coating of TC with anti-EGFR monoclonal antibody cetuximab. Results: Eight stable mCRC lines have been obtained and characterized to confirm their neoplastic origin. Patient NK cells display low capacity to lyse autologous TC (median 12%, range 5%-17%). IL-2 and IL-15 incubation significantly potentiate NK cytotoxic capacity (median18%, range 6%-62% and median 35%, range 28%-65%, respectively). These results refer to an effector:target ratio of 25:1. ADCC assay has shown that cetuximab pre-coating of TC results on an increasing of their susceptibility to NK-mediated lysis. Phenotypical and molecular analysis of activating NK receptors and their ligands present on surface of TC are currently under evaluation. Conclusions: NK cells derived from mCRC patients display measurable cytotoxic activity against autologous primary TC. Susceptibility of NK-mediated lysis is strongly enhanced by pre-incubation of NK cell with cytokines and by pre-coating of TC with cetuximab. These findings, presented upon approval by the study’s data monitoring committee and undergoing confirmation in a larger series, may support a pilot study of NK cells pre-activated ex vivo for treatment of mCRC patients.
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Niu, Haitao, and Laurence Morel. "Intrinsic B cell defect results in abnormal TD antigen response in lupus-prone mice (130.17)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S230—S231. http://dx.doi.org/10.4049/jimmunol.178.supp.130.17.
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Abstract The response to TD antigen NP-KLH was compared between the lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice and control B6 mice. The amount of anti-NP IgG Abs in TC mice was significantly decreased as compared to B6 mice. In contrast, total anti-IgG Abs and anti-dsDNA Abs were increased compared to B6 mice, indicating that the decreased was specific for the immunization. This corresponds to the known poor immunization response of lupus patients. In addition, germinal center (GC) NP-specific B cells were decreased in TC mice compared to B6 mice, although there was no difference in the total number of GC B cells between the two strains. Co-transfer of purified CD43- B and CD4+ T cells from either B6 or TC mice showed that a TC intrinsic B cell defect was involved. B6.Rag−/− mice transferred with non-T BM cells from immunized TC mice lost anti-NP antibody production unlike transfer with BM cells from B6 mice. In contrast, there was similar Ab production between the two strains when transferred with non-T splenocytes, indicating that long-lived plasma cells in TC mice accumulated in spleen, instead of migrating to BM. In conclusion, lupus-prone mice show a deficient response to NP-KLH, in which NP-specific B cells differentiate differently from autoantigen-specific B cells. This deficiency is due at least in part to a defect in B cell maturation and plasma cell migration. SLE susceptibility loci involved in these defects will be mapped in future.
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Koller, A., N. Seyedi, M. E. Gerritsen, and G. Kaley. "EDRF released from microvascular endothelial cells dilates arterioles in vivo." American Journal of Physiology-Heart and Circulatory Physiology 261, no. 1 (July 1, 1991): H128—H133. http://dx.doi.org/10.1152/ajpheart.1991.261.1.h128.
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Microvascular endothelial cells (MECs) from rat epididymal fat pad were isolated and cultured in vitro on Cytodex 3 microcarrier beads. In Krebs-suffused cremaster muscle of pentobarbital-anesthetized rats arteriolar diameters (mean control diam 20.9 +/- 0.9 micron) were measured using image shearing video microscopy. Two lines of suffusate (1.5 ml/min each) were established; one contained a column of microcarrier beads only (no cells in line; NC) the other contained a 1-ml column of MECs grown on beads (through cells; TC). The muscle preparation and the MECs were first treated with indomethacin (Indo; 28 microM). Indo treatment blocked arteriolar dilation to A23187 (1 microM) and arachidonic acid (AA; 0.25 microM) administered into the NC line. A 4.0 +/- 0.6 micron increase in arteriolar diameter was observed, however, when A23187 (but not AA) was infused through the TC line containing Indotreated MECs on beads. The A23187-elicited dilation was abolished by the introduction of NG-monomethyl-L-arginine (L-NMMA; 200 microM) into the TC line. Administration of atropine (2 microM) onto the cremaster muscle via the NC line inhibited the dilations in response to acetylcholine (ACh; 2.7 microM) given through the NC line. Infusion of ACh through the TC line onto the atropine-treated cremaster muscle, however, elicited a 5.8 +/- 1.3 micron increase in arteriolar diameter, a response that was blocked by prior administration of L-NMMA into the TC line. Arteriolar dilation induced by adenosine (0.5 microM) or sodium nitroprusside (0.5 microM) applied via the NC or TC line was unaffected by L-NMMA.(ABSTRACT TRUNCATED AT 250 WORDS)