scholarly journals Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells

2020 ◽  
Vol 3 (8) ◽  
pp. e202000671
Author(s):  
Somsundar V Muralidharan ◽  
Lisa M Nilsson ◽  
Mattias F Lindberg ◽  
Jonas A Nilsson
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Cancer Cells ◽  
Null Allele ◽  
Germ Line ◽  
Protein Levels ◽  
Conditional Allele ◽  
High Level ◽  
Atr Kinase ◽  
Chk1 Kinase

Chk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication. Indeed, Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs, and effects associate with the induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function have relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here, we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germ line. We find that this mouse is overtly normal and does not have problems with erythropoiesis with aging as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated, and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinase-dead Chk1 mouse with a conditional allele, we are able to demonstrate that expression of only one kinase-dead allele, but no wild-type allele, of Chek1 is lethal for Myc-induced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.

scholarly journals Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells

2020 ◽  
Author(s):  
Somsundar V Muralidharan ◽  
Lisa M Nilsson ◽  
Mattias F Lindberg ◽  
Jonas A Nilsson
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Cancer Cells ◽  
Null Allele ◽  
Protein Levels ◽  
Conditional Allele ◽  
Car T ◽  
High Level ◽  
Atr Kinase ◽  
Chk1 Kinase

AbstractChk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication and Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs and effects associates with induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function has relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germline. We find that this mouse is overtly normal and does not have problems with erythropoiesis with ageing as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinase-dead Chk1 mouse with a conditional allele we are able to demonstrate that expression of only one kinase-dead allele, but no wildtype allele, of Chek1 is lethal for Myc-induced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.

scholarly journals To explore immune synergistic function of Quercetin in inhibiting breast cancer cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dan Qiu ◽  
Xianxin Yan ◽  
Xinqin Xiao ◽  
Guijuan Zhang ◽  
Yanqiu Wang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Breast Cancer Cells ◽  
Γδ T Cells ◽  
Killing Effect ◽  
Protein Levels ◽  
Stat1 Signaling ◽  
Mcf 7

Abstract Background The precancerous disease of breast cancer is an inevitable stage in the tumorigenesis and development of breast neoplasms. Quercetin (Que) has shown great potential in breast cancer treatment by inhibiting cell proliferation and regulating T cell function. γδ T cells are a class of nontraditional T cells that have long attracted attention due to their potential in immunotherapy. In this study, we revealed the immunomodulatory function of Que through regulation of the JAK/STAT1 signaling pathway, which was followed by the synergistic killing of breast cancer cells. Methods In the experimental design, we first screened target genes with or without Que treatment, and we intersected the Que target with the disease target by functional enrichment analysis. Second, MCF-10A, MCF-10AT, MCF-7 and MDA-MB-231 breast cancer cell lines were treated with Que for 0 h, 24 h and 48 h. Then, we observed the expression of its subsets by coculturing Que and γδ T cells and coculturing Que and γδ T cells with breast tumor cells to investigate their synergistic killing effect on tumor cells. Finally, Western blotting was used to reveal the changes in proteins related to the JAK/STAT1 signaling pathway after Que treatment in MCF-10AT and MCF-7 cells for 48 h. Results The pathway affected by Que treatment was the JAK/STAT1 signaling pathway and was associated with precancerous breast cancer, as shown by network pharmacology analysis. Que induced apoptosis of MCF-10AT, MCF-7 and MDA-MB-231 cells in a time- and concentration-dependent manner (P < 0.05). Most importantly, Que promoted the differentiation of γδ T cells into the Vδ2 T cell subpopulation. The best ratio of effector cells to target cells (E/T) was 10:1, the killing percentages of γδ T cells against MCF-10A, MCF-10AT, MCF-7, and MDA-MB-231 were 61.44 ± 4.70, 55.52 ± 3.10, 53.94 ± 2.74, and 53.28 ± 1.73 (P = 0.114, P = 0.486, and P = 0.343, respectively), and the strongest killing effect on precancerous breast cancer cells and breast cancer cells was found when the Que concentration was 5 μM and the E/T ratio was 10:1 (64.94 ± 3.61, 64.96 ± 5.45, 55.59 ± 5.98, and 59.04 ± 5.67, respectively). In addition, our results showed that Que increased the protein levels of IFNγ-R, p-JAK2 and p-STAT1 while decreasing the protein levels of PD-L1 (P < 0.0001). Conclusions In conclusion, Que plays a synergistic role in killing breast cancer cells and promoting apoptosis by regulating the expression of IFNγ-R, p-JAK2, p-STAT1 and PD-L1 in the JAK/STAT1 signaling pathway and promoting the regulation of γδ T cells. Que may be a potential drug for the prevention of precancerous breast cancer and adjuvant treatment of breast cancer.

scholarly journals Interleukin-17-Producing CD4+ T Cells Promote Inflammatory Response and Foster Disease Progression in Hyperlipidemic Patients and Atherosclerotic Mice

2021 ◽  
Vol 8 ◽  
Author(s):  
Yin Wang ◽  
Wenming Li ◽  
Tingrui Zhao ◽  
Yao Zou ◽  
Tao Deng ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Th17 Cells ◽  
Early Stage ◽  
Inflammatory Cells ◽  
Chronic Inflammatory Disease ◽  
Interleukin 17 ◽  
Atherosclerotic Lesions ◽  
High Level

Atherosclerosis is a chronic inflammatory disease. Interleukin-17-producing CD4+ T cells (Th17 cells) play important roles in the progression of atherosclerosis. However, most of the studies were focused on the advanced stage of atherosclerosis. In the current study, we investigated the roles of Th17 cells, relevant mechanisms in hyperlipidemic patients, and different stages of atherosclerotic mice. Human blood samples were collected, and percentages of Th17 cells, macrophages, and neutrophils were analyzed by flow cytometry. ApoE−/− mice were fed with high-fat diet (HFD) and sacrificed at different time points to evaluate the infiltration of inflammatory cells at different stages of atherosclerosis. Furthermore, essential mechanisms of IL-17A in atherosclerotic inflammatory milieu formation were studied in vivo by intraperitoneal injection with monoclonal anti-murine IL-17 antibody. Our study reveals the higher percentages of Th17 cells, monocytes, and neutrophils in hyperlipidemic patients compared to healthy donors. Meanwhile, we also identify an infiltration of Th17 cells in the early stage of atherosclerosis (4 weeks after HFD), which maintains at high level until late stage of atherosclerosis (20 weeks after HFD). What is more, inflammatory cells including macrophages and neutrophils were also accumulated in atherosclerotic lesions. Neutralization of IL-17 in ApoE−/− mice resulted in less infiltration of macrophages and neutrophils and smaller atherosclerotic lesions. Importantly, in accordance with what is found in the mouse model, positive correlations between Th17 cells and macrophages or neutrophils were observed in hyperlipidemic patients. In conclusion, our clinical and mouse model data together reveal a pro-atherogenic role of Th17 cells through the promotion of inflammation in hyperlipidemic conditions and different stages of atherosclerosis, which further supports the notion that IL-17 may be a therapy target for the treatment of atherosclerosis.

Immunotherapy of Primary Ovarian Cancer Using Autologous Cytokine Induced Killer Cells Retargeted with Bispecific Antibodies: A Preclinical Study.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2379-2379
Author(s):  
John K. Chan ◽  
Chad A. Hamilton ◽  
Michael K. Cheung ◽  
Mobin Karimi ◽  
Jeanette Baker ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
Mouse Model ◽  
Cancer Cells ◽  
Imaging System ◽  
Bispecific Antibodies ◽  
Ovarian Cancer Cells ◽  
Primary Ovarian Cancer ◽  
Cik Cells

Abstract Cytokine induced killer (CIK) cells are ex-vivo activated and expanded CD8+ natural killer T cells that have been shown to have anti-tumor activity. This is the first study exploring cell killing of primary ovarian carcinoma with and without bispecific antibodies (BSAbs). Primary cancer cells and autologous CIK cells were collected from women with epithelial ovarian cancer. BSAbs against CA125 (BSAbxCA125) and Her2 (BSAbxHer2) were developed using chemical heteroconjugation. On FACS analysis, the expansion and stimulation of CIK cells resulted in a significant increase of CD3+CD8+ and CD3+CD56+ T cells. With enhancement by BSAbs, the mean percent lysis in a 51Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 22% (±0.3) to 89% (±2.1) at an effector to target ratio (E:T) of 100:1. Anti-NKG2D antibodies significantly attenuated the CIK activity by 57% on primary cells. In a xenograft SCID mouse model, real-time tumor regression and progression was visualized using a non-invasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8+NKG2D+ CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 (p=0.0002) and BSAbxHer2 (p=0.0002) had a significant reduction in tumor burden and improvement in survival versus those treated with CIK cells alone (p=0.03). BSAbs significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis appears to be mediated in part by the NKG2D receptor.

Resident memory and recirculating memory T cells cooperate to maintain disease in a mouse model of vitiligo

immuneACCESS ◽  
2018 ◽  
Author(s):  
JM Richmond ◽  
JP Strassner ◽  
M Rashighi ◽  
P Agarwal ◽  
M Garg ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Memory T Cells

scholarly journals Cross-presentation of a TAP-independent signal peptide induces CD8 T immunity to escaped cancers but necessitates anchor replacement

2021 ◽  
Author(s):  
Koen A. Marijt ◽  
Lisa Griffioen ◽  
Laura Blijleven ◽  
Sjoerd. H. van der Burg ◽  
Thorbald van Hall
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Cd8 T Cells ◽  
Antigen Processing ◽  
Signal Sequence ◽  
Cell Repertoire ◽  
Cross Presentation ◽  
Normal Tissues ◽  
Cell Immunity

AbstractCancer cells frequently display defects in their antigen-processing pathway and thereby evade CD8 T cell immunity. We described a novel category of cancer antigens, named TEIPP, that emerge on cancers with functional loss of the peptide pump TAP. TEIPPs are non-mutated neoantigens despite their ‘self’ origin by virtue of their absence on normal tissues. Here, we describe the development of a synthetic long peptide (SLP) vaccine for the most immunogenic TEIPP antigen identified thus far, derived from the TAP-independent LRPAP1 signal sequence. LRPAP121–30-specific CD8 T cells were present in blood of all tested healthy donors as well as patients with non-small cell lung adenocarcinoma. SLPs with natural flanking, however, failed to be cross-presented by monocyte-derived dendritic cells. Since the C-terminus of LRPAP121–30 is an unconventional and weakly binding serine (S), we investigated if replacement of this anchor would result in efficient cross-presentation. Exchange into a valine (V) resulted in higher HLA-A2 binding affinity and enhanced T cell stimulation. Importantly, CD8 T cells isolated using the V-variant were able to bind tetramers with the natural S-variant and respond to TAP-deficient cancer cells. A functional screen with an array of N-terminal and C-terminal extended SLPs pointed at the 24-mer V-SLP, elongated at the N-terminus, as most optimal vaccine candidate. This SLP was efficiently cross-presented and consistently induced a strong polyclonal LRPAP121–30-specific CD8 T cells from the endogenous T cell repertoire. Thus, we designed a TEIPP SLP vaccine from the LRPAP1 signal sequence ready for validation in clinical trials.

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