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.
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Project description:Cytotoxic T lymphocytes (CTLs) are important in controlling some viral infections, and therapies involving the transfer of large numbers of cancer-specific CTLs have been successfully used to treat several types of cancers in humans. While the molecular mechanisms of how CTLs kill their targets are relatively well understood, we still lack a solid quantitative understanding of the kinetics and efficiency by which CTLs kill their targets in vivo. Collagen-fibrin-gel-based assays provide a tissue-like environment for the migration of CTLs, making them an attractive system to study T cell cytotoxicity in in vivo-like conditions. Budhu.et al. systematically varied the number of peptide (SIINFEKL)-pulsed B16 melanoma cells and SIINFEKL-specific CTLs (OT-1) and measured the remaining targets at different times after target and CTL co-inoculation into collagen-fibrin gels. The authors proposed that their data were consistent with a simple model in which tumors grow exponentially and are killed by CTLs at a per capita rate proportional to the CTL density in the gel. By fitting several alternative mathematical models to these data, we found that this simple "exponential-growth-mass-action-killing" model did not precisely describe the data. However, determining the best-fit model proved difficult because the best-performing model was dependent on the specific dataset chosen for the analysis. When considering all data that include biologically realistic CTL concentrations (E≤107cell/mL), the model in which tumors grow exponentially and CTLs suppress tumor's growth non-lytically and kill tumors according to the mass-action law (SiGMA model) fit the data with the best quality. A novel power analysis suggested that longer experiments (∼3-4 days) with four measurements of B16 tumor cell concentrations for a range of CTL concentrations would best allow discriminating between alternative models. Taken together, our results suggested that the interactions between tumors and CTLs in collagen-fibrin gels are more complex than a simple exponential-growth-mass-action killing model and provide support for the hypothesis that CTLs' impact on tumors may go beyond direct cytotoxicity.
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Project description:Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.
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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.
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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.
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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.
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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.
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Project description:CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. In a previous study, we identified a subset of murine effector CTLs expressing the hepatocyte growth factor (HGF) receptor, c-Met (c-Met+ CTLs), that are endowed with enhanced cytolytic capacity. HGF directly inhibited the cytolytic function of c-Met+ CTLs, both in 2D in vitro assays and in vivo, leading to reduced T cell responses against metastatic melanoma. To further investigate the role of c-Met+ CTLs in a three-dimensional (3D) setting, we studied their function within B16 melanoma spheroids and examined the impact of cell-cell contact on the modulation of inhibitory checkpoint molecules' expression, such as KLRG1, PD-1, and CTLA-4. Additionally, we evaluated the cytolytic capacity of human CTL clones expressing c-Met (c-Met+) and compared it to c-Met- CTL clones. Our results indicated that, similar to their murine counterparts, c-Met+ human CTL clones exhibited increased cytolytic activity compared to c-Met- CTL clones, and this enhanced function was negatively regulated by the presence of HGF. Taken together, our findings highlight the potential of targeting the HGF/c-Met pathway to modulate CTL-mediated anti-tumor immunity. This research holds promise for developing strategies to enhance the effectiveness of CTL-based immunotherapies against cancer.
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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.
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Project description:Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here, we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hr of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hr after tumor cell arrival, but after 24 hr of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24-hr period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing, the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung.
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