Dynamic visualization of the whole process of cytotoxic T lymphocytes killing B16 tumor cells in vitro.
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ABSTRACT:
SUBMITTER: Qi S
PROVIDER: S-EPMC6992963 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Major histocompatibility complex (MHC)-unrestricted cytotoxic lymphocytes (CLs) such as natural killer (NK) cells can detect and destroy tumor and virus-infected cells resistant to T cell-mediated killing. Here, we performed genome-wide genetic screens to identify tumor-intrinsic genes regulating killing by MHC-unrestricted CLs. A group of genes identified in our screens encode enzymes for the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is not involved in tumor response to T cell-mediated cytotoxicity. Another gene identified in the screens was PBRM1, which encodes a subunit of the PBAF form of the SWI/SNF chromatin-remodeling complex. PBRM1 mutations in tumor cells cause resistance to MHC-unrestricted killing, in contrast to their sensitizing effects on T cell-mediated killing. PBRM1 and the GPI biosynthetic pathway regulate the ligands of NK cell receptors in tumor cells and promote cytolytic granule secretion in CLs. The regulators identified in this work represent potential targets for cancer immunotherapy.
| S-EPMC7695474 | biostudies-literature
Project description:Cytotoxic T lymphocytes (CTL) help control virus infections by killing virus-infected (target) cells. How fast do CTL find and kill target cells in vivo? Experiments allow us to follow populations of labeled target cells after their transfer into immune mice where they are killed by CTL. Here, we develop models of the migration and killing processes involved in these experiments and use them to quantitatively analyze the in vivo killing data. These models allow us to estimate the rate constant for killing of target cells by CTL.
| S-EPMC1785271 | biostudies-literature
Project description:Glioblastoma multiforme, the most aggressive primary brain tumor, is maintained by a subpopulation of glioma cells with self-renewal properties that are able to recapitulate the entire tumor even after surgical resection or chemo-radiotherapy. This typifies the vast heterogeneity of this tumor with the two extremes represented on one end by the glioma stemlike cells (GSC) and on the other by the glioma differentiated cells (GDC). Interestingly, GSC are more sensitive to immune effector cells than the GDC counterpart. However, how GSC impact on the killing on the GDC and vice versa is not clear. Using a newly developed cytotoxicity assay allowing to simultaneously monitor cytotoxic lymphocytes-mediated killing of GSC and GDC, we found that although GSC were always better killed and that their presence enhanced the killing of GDC. In contrast, an excess of GDC had a mild protective effect on the killing of GSC, depending on the CTL type. Overall, our results suggest that during combination therapy, immunotherapy would be the most effective after prior treatment with conventional therapies.
| S-EPMC4830556 | biostudies-literature
Project description:Cytotoxic T lymphocyte (CTL)-mediated killing involves the formation of a synapse with a target cell, followed by delivery of perforin and granzymes. Previously, we derived a general functional response for CTL killing while considering that CTLs form stable synapses (i.e., single-stage) and that the number of conjugates remains at steady state. However, the killing of target cells sometimes requires multiple engagements (i.e., multistage). To study how multistage killing and a lack of steady state influence the functional response, we here analyze a set of differential equations as well as simulations employing the cellular Potts model, in both cases describing CTLs that kill target cells. We find that at steady state the total killing rate (i.e., the number of target cells killed by all CTLs) is well described by the previously derived double saturation function. Compared to single-stage killing, the total killing rate during multistage killing saturates at higher CTL and target cell densities. Importantly, when the killing is measured before the steady state is approached, a qualitatively different functional response emerges for two reasons: First, the killing signal of each CTL gets diluted over several targets and because this dilution effect is strongest at high target cell densities; this can result in a peak in the dependence of the total killing rate on the target cell density. Second, the total killing rate exhibits a sigmoid dependence on the CTL density when killing is a multistage process, because it takes typically more than one CTL to kill a target. In conclusion, a sigmoid dependence of the killing rate on the CTLs during initial phases of killing may be indicative of a multistage killing process. Observation of a sigmoid functional response may thus arise from a dilution effect and is not necessarily due to cooperative behavior of the CTLs.
| S-EPMC5375173 | biostudies-literature
Project description:The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell-mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.
| S-EPMC2212464 | biostudies-other
Project description:Cytotoxic T lymphocytes (CTLs) are important agents in the control of intracellular pathogens, which specifically recognize and kill infected cells. Recently developed experimental methods allow the estimation of the CTL's efficacy in detecting and clearing infected host cells. One method, the in vivo killing assay, utilizes the adoptive transfer of antigen displaying target cells into the bloodstream of mice. Surprisingly, killing efficacies measured by this method are often much higher than estimates obtained by other methods based on, for instance, the dynamics of escape mutations. In this study, we investigated what fraction of this variation can be explained by differences in peptide loads employed in in vivo killing assays. We addressed this question in mice immunized with lymphocytic choriomeningitis virus (LCMV). We conducted in vivo killing assays varying the loads of the immunodominant epitope GP33 on target cells. Using a mathematical model, we determined the efficacy of effector and memory CTL, as well as CTL in chronically infected mice. We found that the killing efficacy is substantially reduced at lower peptide loads. For physiological peptide loads, our analysis predicts more than a factor 10 lower CTL efficacies than at maximum peptide loads. Assuming that the efficacy scales linearly with the frequency of CTL, a clear hierarchy emerges among the groups across all peptide antigen concentrations. The group of mice with chronic LCMV infections shows a consistently higher killing efficacy per CTL than the acutely infected mouse group, which in turn has a consistently larger efficacy than the memory mouse group. We conclude that CTL killing efficacy dependence on surface epitope frequencies can only partially explain the variation in in vivo killing efficacy estimates across experimental methods and viral systems, which vary about four orders of magnitude. In contrast, peptide load differences can explain at most two orders of magnitude.
| S-EPMC4416789 | biostudies-literature
Project description:Despite the remarkable progress of adoptive T cell therapy in cancer treatment, there remains an urgent need for the noninvasive tracking of the transfused T cells in patients to determine their biodistribution, viability, and functionality. With emerging molecular imaging technologies and cell-labeling methods, noninvasive in vivo cell tracking is experiencing impressive progress toward revealing the mechanisms and functions of these cells in real time in preclinical and clinical studies. Such cell tracking methods have an important role in developing effective T cell therapeutic strategies and steering decision-making process in clinical trials. On the other hand, they could provide crucial information to accelerate the regulatory approval process on the T cell therapy. In this review, we revisit the advances in tracking the tumor-specific CTLs, highlighting the latest development in human studies and the key challenges.
| S-EPMC4142291 | biostudies-other
Project description:BackgroundPI3K? is a lipid kinase of the phosphoinositide 3-kinase class 1A family and involved in early signaling events of leukocytes regulating proliferation, differentiation and survival. Currently, several inhibitors of PI3K? are under investigation for the treatment of hematopoietic malignancies. In contrast to the beneficial effect of inhibiting PI3K? in tumor cells, several studies reported the requirement of PI3K? for the function of immune cells, such as natural killer and T helper cells. Cytotoxic T lymphocytes (CTLs) are essential for tumor surveillance. The scope of this study is to clarify the potential impact of PI3K? inhibition on the function of CTLs with emphasis on tumor surveillance.Principal findingsPI3K?-deficient mice develop significantly bigger tumors when challenged with MC38 colon adenocarcinoma cells. This defect is accounted for by the fact that PI3K? controls the secretory perforin-granzyme pathway as well as the death-receptor pathway of CTL-mediated cytotoxicity, leading to severely diminished cytotoxicity against target cells in vitro and in vivo in the absence of PI3K? expression. PI3K?-deficient CTLs express low mRNA levels of important components of the cytotoxic machinery, e.g. prf1, grzmA, grzmB, fasl and trail. Accordingly, PI3K?-deficient tumor-infiltrating CTLs display a phenotype reminiscent of naïve T cells (CD69(low)CD62L(high)). In addition, electrophysiological capacitance measurements confirmed a fundamental degranulation defect of PI3K?-/- CTLs.ConclusionOur results demonstrate that CTL-mediated tumor surveillance is severely impaired in the absence of PI3K? and predict that impaired immunosurveillance may limit the effectiveness of PI3K? inhibitors in long-term treatment.
| S-EPMC3396622 | biostudies-literature
Project description:Recent studies have proposed that tumor-specific tumor-infiltrating CD8+ T lymphocytes (CD8 TIL) can be classified into two main groups: "exhausted" TILs, characterized by high expression of the inhibitory receptors PD-1 and TIM-3 and lack of transcription factor 1 (Tcf1); and "memory-like" TILs, with self-renewal capacity and co-expressing Tcf1 and PD-1. However, a comprehensive definition of the heterogeneity existing within CD8 TILs has yet to be clearly established. To investigate this heterogeneity at the transcriptomic level, we performed paired single-cell RNA and TCR sequencing of CD8 T cells infiltrating B16 murine melanoma tumors, including cells of known tumor specificity. Unsupervised clustering and gene-signature analysis revealed four distinct CD8 TIL states - exhausted, memory-like, naïve and effector memory-like (EM-like) - and predicted novel markers, including Ly6C for the EM-like cells, that were validated by flow cytometry. Tumor-specific PMEL T cells were predominantly found within the exhausted and memory-like states but also within the EM-like state. Further, T cell receptor sequencing revealed a large clonal expansion of exhausted, memory-like and EM-like cells with partial clonal relatedness between them. Finally, meta-analyses of public bulk and single-cell RNA-seq data suggested that anti-PD-1 treatment induces the expansion of EM-like cells. Our reference map of the transcriptomic landscape of murine CD8 TILs will help interpreting future bulk and single-cell transcriptomic studies and may guide the analysis of CD8IL subpopulations in response to therapeutic interventions.
| S-EPMC7153840 | biostudies-literature
Project description:The present study investigated whether antigen presentation of the Oct4 and Sox2 peptides by CD154-activated B lymphocytes can enhance the killing effect of CD8+ cytotoxic T lymphocytes (CTLs) on lung stem-like cancer cells (SLCCs). The CTLs were generated using an accelerated co-cultured dendritic cells (DC) (acDC) assay by incubating human peripheral blood mononuclear cells (PBMCs) from non-small-cell lung cancer patients with antigen peptides of Oct4 and Sox2 in the presence of several DC-activating agents. CD154+ NIH3T3 cells prepared by CD154 lentiviral transfection were used as feeder layer to activate primary B cells (CD19+) obtained from PBMCs. Activated B cells were co-cultured with CTLs to present antigen peptides of Oct4 and Sox2. CTLs co-cultured with activated B cells were evaluated for the levels of secreted inflammatory cytokines using ELISA. In addition, the killing effect of the CTLs on SLCCs derived from cisplatin-resistant strain of human lung cancer cell line PC9 was evaluated by flow cytometry using CFSE labeling of the target cells. After the acDC assay, the PBMCs exhibited a significant (p<0.01) increase in the population of CD8+/CD3+ cells, indicating successful preparation of CTLs. The primary B cells cultured on the CD154+ NIH3T3 feeder layer resulted in significant (p<0.01) increase in the proportions of population expressing CD80, CD86, or HLA-A, indicating successful activation of the B cells. The co-culture of CTLs with CD154-activated B cells presenting the Oct4 and Sox2 peptides caused significant increase in the levels of secretory inflammatory cytokines and exhibited enhanced killing of the SLCCs derived from cisplatin-resistant PC9 cells. Antigen presentation of the Oct4 and Sox2 peptides by CD154-activated B cells can enhance the killing effect of CTLs towards lung SLCCs.
| S-EPMC5771344 | biostudies-literature