Cytotoxic T Lymphocytes Migration Research Articles

Cancer-on-a-chip model shows that the adenomatous polyposis coli mutation impairs T cell engagement and killing of cancer spheroids

Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance, to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the 2 h preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction.

Cytotoxic T Lymphocytes Control Growth of B16 Tumor Cells in Collagen-Fibrin Gels by Cytolytic and Non-Lytic Mechanisms.

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.

CTLs From Patients With Atherosclerosis Show Elevated Adhesiveness and Distinct Integrin Expression Patterns on 2D Substrates.

Atherosclerosis is the major cause of cardiovascular disease that is characterized by plaque formation in the blood vessel wall. Atherosclerotic plaques represent sites of chronic inflammation with diverse cell content that is shifted toward the prevalence of cytotoxic T-lymphocytes (CTLs) upon plaque progression. The studies of CTL recruitment to atherosclerotic plaques require adequate in vitro models accounting for CTL interactions with chemokine-ligands and extracellular matrix fibers via surface chemokine receptors and integrins. Here we applied such a model by investigating CTL adhesion and migration on six types of coated surfaces. We assessed adhesion and motility metrics, the expression of chemokine receptors, and integrins in CTLs of patients with atherosclerosis and healthy donors. Using fibronectin, platelet-poor plasma from patients with atherosclerosis, and conditioned medium from atherosclerotic plaques we revealed the role of substrate in CTL adhesiveness: fibronectin alone and fibronectin combined with platelet-poor plasma and conditioned medium elevated the CTL adhesiveness – in patients the elevation was significantly higher than in healthy donors (p = 0.02, mixed 2-way ANOVA model). This was in line with our finding that the expression levels of integrin-coding mRNAs were elevated in the presence of fibronectin (p < 0.05) and ITGB1, ITGA1, and ITGA4 were specifically upregulated in patients compared to healthy donors (p < 0.01). Our experimental model did not affect the expression levels of mRNAs CCR4, CCR5, and CX3CR1 coding the chemokine receptors that drive T-lymphocyte migration to plaques. Thus, we demonstrated the substrate-dependence of integrin expression and discriminated CTLs from patients and healthy donors by adhesion parameters and integrin expression levels.

Macroscopic limit of a kinetic model describing the switch in T cell migration modes via binary interactions

Experimental results on the immune response to cancer indicate that activation of cytotoxic T lymphocytes (CTLs) through interactions with dendritic cells (DCs) can trigger a change in CTL migration patterns. In particular, while CTLs in the pre-activation state move in a non-local search pattern, the search pattern of activated CTLs is more localised. In this paper, we develop a kinetic model for such a switch in CTL migration modes. The model is formulated as a coupled system of balance equations for the one-particle distribution functions of CTLs in the pre-activation state, activated CTLs and DCs. CTL activation is modelled via binary interactions between CTLs in the pre-activation state and DCs. Moreover, cell motion is represented as a velocity-jump process, with the running time of CTLs in the pre-activation state following a long-tailed distribution, which is consistent with a Lévy walk, and the running time of activated CTLs following a Poisson distribution, which corresponds to Brownian motion. We formally show that the macroscopic limit of the model comprises a coupled system of balance equations for the cell densities, whereby activated CTL movement is described via a classical diffusion term, whilst a fractional diffusion term describes the movement of CTLs in the pre-activation state. The modelling approach presented here and its possible generalisations are expected to find applications in the study of the immune response to cancer and in other biological contexts in which switch from non-local to localised migration patterns occurs.

High Glucose Enhances Cytotoxic T Lymphocyte-Mediated Cytotoxicity.

Cytotoxic T lymphocytes (CTLs) are key players to eliminate tumorigenic or pathogen-infected cells using lytic granules (LG) and Fas ligand (FasL) pathways. Depletion of glucose leads to severely impaired cytotoxic function of CTLs. However, the impact of excessive glucose on CTL functions still remains largely unknown. Here we used primary human CD8+ T cells, which were stimulated by CD3/CD28 beads and cultured in medium either containing high glucose (HG, 25 mM) or normal glucose (NG, 5.6 mM). We found that in HG-CTLs, glucose uptake and glycolysis were enhanced, whereas proliferation remained unaltered. Furthermore, CTLs cultured in HG exhibited an enhanced CTL killing efficiency compared to their counterparts in NG. Unexpectedly, expression of cytotoxic proteins (perforin, granzyme A, granzyme B and FasL), LG release, cytokine/cytotoxic protein release and CTL migration remained unchanged in HG-cultured CTLs. Interestingly, additional extracellular Ca2+ diminished HG-enhanced CTL killing function. Our findings suggest that in an environment with excessive glucose, CTLs could eliminate target cells more efficiently, at least for a certain period of time, in a Ca2+-dependent manner.

Quantitative evaluation of tumor-specific T cells in tumors and lymphoid tissues.

Homing of tumor-specific cytotoxic T lymphocytes (CTLs) to the tumor tissues represents a vital step in procuring an effective anti-tumor immune response. Intratumoral accumulation of tumor-specific CTLs can be supported through local chemokine modulation using immune adjuvants or viral vectors, as well as vaccination, using peptide, protein or cell-based vaccines, including dendritic cell (DC) vaccines. Clinical and pre-clinical studies demonstrate that the current immunotherapy regimens are only effective when high numbers of CTLs are present within the tumor microenvironment (TME). Notably, many types of cancer take advantage of this principle and restrict T cell migration into the tumor, subverting the anti-tumor immune response and allowing uncontrolled tumor growth. This chapter discusses the mechanisms involved in the migration of CTLs into tumors and describes the feasible method of evaluating treatment-induced changes in the numbers of polyclonal tumor-specific CTLs in the TME and lymphoid tissues. The described method is widely applicable to multiple tumor models with wild-type antigen expression patterns, without the need for genetically-manipulated cancer cells or animals expressing defined T cell receptors.

Hedgehog signaling induces PDL‐1 expression and subsequently inactivates effector T cell function and promotes gastric cancer cell proliferation

BackgroundHedgehog (Hh) signaling plays a crucial role in tumor growth and chemoresistance in a variety of tumors including gastric cancer. Tumor cells often express elevated programmed cell death 1 (PD‐L1) that interacts with PD‐1 on CD8+ cytotoxic T lymphocytes (CTLs). This interaction inhibits CTL effector function, leading to immune evasion and cancer cell proliferation. Regulation of PD‐L1 may be a promising therapeutic strategy, but the regulatory mechanism remains unclear in gastric cancer.HypothesisHh signaling induces PDL‐1 expression and subsequently inactivates effector T cell function and promotes gastric cancer cell proliferation.MethodsMouse‐derived gastric cancer organoids were generated from a triple‐transgenic model engineered to express an activated GLI2 allele, GLI2A, in Lgr5‐expressing stem cells when treated with doxycycline (iLgr5;GLI2A mTGOs). Organoids were also derived from normal mouse gastric tissue (mGOs). Conditioned media was collected from mTGOs (mTGOCM) and mGOs (mGOCM). Bone marrow‐derived dendritic cells (DCs) were pulsed with either mGOCM or mTGOCM for 24 hrs and co‐cultured with CTLs for 48 hrs. Pulsed DCs and CTLs were then co‐cultured with either mGOs or mTGOs for 24 hrs in presence or absence of PD‐L1 inhibitor (PDL‐1I). Apoptosis and cell proliferation were measured using TUNEL or Edu Imaging Assay Kits respectively. mTGOs were treated with GANT61. PD‐L1 expression and cell proliferation were measured after 48 hrs. iLgr5;GLI2A mice were injected with either Hh inhibitor GANT61 or phosphate buffered saline for 20 days and stomach tissues were immunostained for PD‐L1 and metaplastic markers CD44v9 and lectin Griffonia simplicifolia (GSII). Human‐derived gastric cancer organoids (huTGO) were derived from resected tumors and analyzed for Gli2 and PD‐L1 expression. HuTGOs were treated for 48 hours with gastric cancer chemotherapeutic agents (Epirubicin, Oxaliplatin, and 5‐FU) in presence or absence of GANT61 and organoid proliferation measured by MTS proliferation assay.Results Increased Gli‐2 correlates with the elevated PD‐L1 expression: ILgr5;GLI2A mice developed rapid gastric adenocarcinoma within the antrum. Organoids derived from these tumors (mTGOs) expressed Gli2 that correlated with elevated PD‐L1 expression when compared to mGOs. Inhibition of PD‐L1 in mTGO/immune cell co‐cultures induces organoid death: CTLs co‐cultured with DCs pulsed with mTGOCM had increased expression of PD‐1, IFNg, and IL‐2, compared to co‐cultures using unpulsed DCs or DCs pulsed with mGOCM. Live time‐lapse microscopy revealed migration of DCs and CTLs toward mTGOs in co‐culture. Pulsed DC/CTL/mTGO co‐cultures revealed no apoptosis or organoid death. Interestingly, when pulsed DC/CTL/mTGO co‐cultures were treated with PDL‐1I, there was induced organoid apoptosis and death. Hedgehog signaling regulates PD‐L1 expression and organoid proliferation: Hh inhibitor, GANT‐61 significantly reduced PD‐L1 expression and tumor cell proliferation both in vitro in mTGOs and in vivo in ILgr5;GLI2A mice. In vivo, GANT61 treatment resulted in decreased PD‐L1 expression that correlated with loss of tumor growth and decreased metaplasia. Inhibition of Hedgehog signaling sensitizes organoids to chemotherapy: Chemotherapy‐induced cell death in Gli2/PD‐L1+ve huTGOs pretreated with GANT61. ConclusionHh signaling plays a role as a regulator of PDL‐1 expression in gastric cancer cells to promote tumor growth.Support or Funding Information1RO1DK083402 (Zavros RO1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A calcium optimum for cytotoxic T lymphocyte and natural killer cell cytotoxicity.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca2+ ]i (between 122 and 334nm) and [Ca2+ ]o (between 23 and 625μm) for efficient cancer cell elimination, well below blood plasma Ca2+ levels. As predicted from these results, partial down-regulation of the Ca2+ channel Orai1 in CTLs paradoxically increases perforin-dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca2+ optimum compatible with this low Ca2+ optimum for efficient cancer cell killing, whereas the Ca2+ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca2+ ]o . We propose that a partial inhibition of Ca2+ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to protect the human body against cancer. Ca2+ is a key metabolic factor for lymphocyte function and cancer homeostasis. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity against cancer cells and found that CTLs have a bell-shaped Ca2+ dependence with an optimum for cancer cell elimination at rather low [Ca2+ ]o (23-625μm) and [Ca2+ ]i (122-334nm). This finding predicts that a partial inhibition of Orai1 should increase (rather than decrease) cytotoxicity of CTLs at [Ca2+ ]o higher than 625μm. We tested this hypothesis in CTLs and indeed found that partial down-regulation of Orai1 by siRNA increases the efficiency of cancer cell killing. We found two mechanisms that may account for the Ca2+ optimum of cancer cell killing: (1) migration velocity and persistence have a moderate optimum between 500 and 1000μm [Ca2+ ]o in CTLs, and (2) lytic granule release at the immune synapse between CTLs and cancer cells is increased at 146μm compared to 3 or 800μm, compatible with the Ca2+ optimum for cancer cell killing. It has been demonstrated in many cancer cell types that Orai1-dependent Ca2+ signals enhance proliferation. We propose that a decrease of [Ca2+ ]o or partial inhibition of Orai1 activity by selective blockers in the tumour microenvironment could efficiently reduce cancer growth by simultaneously increasing CTL and NK cell cytotoxicity and decreasing cancer cell proliferation.

Marek’s disease virus immunosuppression alters host cellular responses and immune gene expression in the skin of infected chickens

Marek’s disease virus (MDV), a highly cell-associated lymphotropic α-herpesvirus, is the causative agent of Marek’s disease (MD) in domestic chickens. MDV replicates in chicken lymphocytes and establishes a latent infection within CD4+ T cells. The latently infected CD4+ T cells carry the virus to visceral organs, peripheral nerves, and feather follicle epithelium (FFE). FFE is the only anatomical site where infectious enveloped cell-free virus particles are produced and disseminated into the environment. This study investigated the immunological responses and mechanism of viral-induced immunosuppression and immune evasion in the FFE. Strong viral replication and lack of a significant number of cytotoxic T lymphocytes (CTL) in the infected tissues was prominent. Although the overall gene expression pattern was suggestive of a Th1 type immune response, the expression levels of several key immune genes were down regulated in the infected tissues. The mechanism of MDV-induced immunosuppression appears to be through inhibition of CTL function due to down regulation of CD8 glycoprotein and/or blocking of CTL migration due to decrease expression of cell adhesion molecules.

Leupaxin Regulates LFA-1 Related Function in Cytotoxic T Lymphocytes (CTL)

Abstract Background Leupaxin is an adaptor protein that functions downstream of integrin receptor engagement. It contains N-terminal LD domains and C-terminal LIM domains. Although leupaxin is primarily expressed in leukocytes, it is very little known about the localization and function in leukocytes. Results Degranulation follows the formation of the immunological synapse (IS) and recruitment of MTOC to the IS. Leupaxin co-localizes with MTOC and microtubules, and is recruited to the IS during CTL degranulation. Through deletion of leupaxin domains, it shows that both LD2 and LD4 domains are sufficient for leupaxin recruitment to the IS. Leupaxin fails to be recruited to the IS after deleting LD2–4 domains. Leupaxin is a focal adhesion protein in adherent cells. Leukocytes do not form classical focal adhesions during migration. We have found that leupaxin is recruited to the contact zone during CTL migration on ICAM-1, and forms very dynamic focal adhesion (FA)-like structures. These structures are assembled in the leading edge, and disappear in the trailing edge. Leupaxin also co-localizes with vinculin and talin at the FA-like structures, which are both FA proteins. Different from the role of leupaxin at the IS, leupaxin C-terminal LIM domain contributes to the localization of leupaxin at the FA-like structures. Conclusions Leupaxin localization suggests that it contributes to LFA-1 related processes in CTL, including degranulation and migration, but with different domains. Leupaxin LD domains are necessary for the recruitment to the IS during degranulation, whereas LIM domains are necessary for the formation of FA-like structures during migration.

Requirement of interleukin 7 signaling for anti-tumor immune response under lymphopenic conditions in a murine lung carcinoma model.

Induction of lymphopenia before adoptive transfer of T cells was followed by lymphopenia-induced proliferation (LIP) and generated a potent anti-tumor immune response in rodents and in a clinical setting. Previously, we reported that CD28 signaling is essential for the differentiation of functional effector cytotoxic T lymphocytes (CTLs) under lymphopenic conditions and sequential LIP of T cells. In this study, to clarify the correlation between LIP and the anti-tumor effect, LIP was inhibited with interleukin 7 (IL7) receptor blockade at various stages, and the anti-tumor effect then assessed. We confirmed that IL7 signaling at the start of LIP is crucial for the anti-tumor immune response. In contrast, continuous IL7 signaling was not required for tumor regression, although LIP of naïve CD8+ T cells is usually regulated by IL7. The expansion and migration of CTLs in lymphopenic hosts depend on IL7 signaling during the induction phase. Here, we propose that IL7 signaling and subsequent LIP of T cells have distinct roles in the induction of T cell immunity during lymphopenia.

Modeling Immune Cell Migration

Cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells are the killer cells of the immune system and are responsible for the elimination of potential dangers, such as virus-infected and tumor cells. CTLs and NK cells migrate actively within tissues to encounter target cells, and form a tight connection with them called the immunological synapse that enables killing. However, how migration is regulated in these cells for an efficient reach of their target remains a central question in immunology. Here we analyzed the migration of CTLs and NK cells in 2 dimensions in the absence of external cues to mirror the initial immune response where no signals guide killer cells to their target. By calculating the mean square displacement and the velocity autocorrelation we found that CTLs perform a persistent random walk. Furthermore, cell shape analysis enabled us to establish a model for killer cell migration where the movement of the cell is determined by several independent internal “force generators”. Each of them switches between active and inactive states. We found a correlation between the number of the “force generators” and the duration of their activity to the number and life time of lamellipodia. Our numerical simulations revealed that the search time in a given space depends on the behavior of cells upon contact with boundaries. We believe that simulation of migration and search strategy of killer cells will ultimately lead to an improved understanding of the immune response.

Abstract PR04: Intravital imaging of cytolytic activity reveals how poor tumor vascularization and hypoxia limit tumor rejection

Abstract Background: Immunotherapies that harness cytotoxic T lymphocytes (CTLs) to eradicate solid tumors, melanoma in particular, have re-emerged as potent clinical options in recent years. Improving the dispersion and cytotoxicity of intratumoral CTLs may further benefit patients. One of the obstacles to successful treatment is thought to be poor vascularization of tumors, which leads to sub-optimal tumor infiltration. In contrast, the effects of disrupted vascularization and the hypoxia it introduces on CTL migration and function within tumors have not been investigated. Methods: To study whether tumor vascularity can shape anti-tumor CTL responses, we combined immunohistochemistry of human melanoma samples with dynamic two-photon imaging within B16 melanoma tumors expressing ovalbumin (OVA) in live mice. Results: In patients, CD8 T cells were twofold denser around peripheral vessels than around deep vessels, and fourfold denser than in avascular areas. This pattern was clearest in poorly-infiltrated tumors. Correspondingly, in mice, adoptively transferred OT-I CTLs, specific for OVA, preferentially infiltrated B16-OVA tumors through peripheral ICAM1+ vessels. CTLs remained initially at their entry areas, crawling vigorously only within 50µm of flowing blood vessels. These dense CTLs swarms efficiently cleared perivascular areas of tumor cells but left avascular areas intact. In contrast, Foxp3+ regulatory T cells could venture farther away from blood vessels. Target killing was antigen-specific, contact-dependent and perforin-mediated, and required multiple CTLs to engage one target cell to be effective. Several findings converge to suggest that perivascular CTL confinement is oxygen-dependent: occlusion of intratumoral blood vessels resulted in instantaneous and reversible arrest of CTLs. Immunohistology indicated that CTLs avoided hypoxic tumor areas. Hypoxia and mitochondrial uncoupling interfered with CTL migration in vitro and, finally, highly vascular tumors grown in a matrix containing the pro-angiogenic factor bFGF were more easily rejected after CTL injection, displayed delayed growth in untreated mice but were not affected in immunodeficient mice. Although such normalized tumors attracted normal numbers of CTLs, these cells migrated more freely within the tumors. Conclusions: We propose that CTLs depend on oxygen for intratumoral locomotion and cytolysis. Incomplete vascularization promotes patchy infiltration and tumor escape; tumor immunotherapy may thus benefit from an extensive and functional vasculature. Citation Format: Guy Shakhar, Yoav Manaster, Tali Feferman. Intravital imaging of cytolytic activity reveals how poor tumor vascularization and hypoxia limit tumor rejection. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr PR04.

Midline 1 controls polarization and migration of murine cytotoxic T cells.

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2 A levels. Loss-of-function mutations in MID1 lead to the human X-linked Opitz G/BBB (OS) syndrome characterized by defective midline development during embryogenesis. We have recently shown that MID1 is strongly up-regulated in murine cytotoxic T lymphocytes (CTLs), and that it has a significant impact on exocytosis of lytic granules and the killing capacity of CTLs. The aims of the present study were to determine the localization of MID1 in migrating CTLs, and to investigate whether MID1 affects CTL polarization and migration. We found that MID1 mainly localizes to the uropod of migrating CTLs and that it has a substantial impact on CTL polarization and migration in vitro. Furthermore, analysis of contact hypersensitivity responses supported that MID1 controls effector functions of CTLs in hapten-challenged skin in vivo. These results provide significant new knowledge on the role of MID1 in CTL biology.

A Mathematical Model of Immune-System-Melanoma Competition

We present a mathematical model developed to reproduce the immune response entitled with the combined administration of activated OT1 cytotoxic T lymphocytes (CTLs) and Anti-CD137 monoclonal antibodies. The treatment is directed against melanoma in B16 OVA mouse models exposed to a specific immunotherapy strategy. We model two compartments: the injection point compartment where the treatment is administered and the skin compartment where melanoma tumor cells proliferate. To model the migration of OT1 CTLs and antibodies from the injection to the skin compartment, we use delay differential equations (DDEs). The outcomes of the mathematical model are in good agreement with the in vivo results. Moreover, sensitivity analysis of the mathematical model underlines the key role of OT1 CTLs and suggests that a possible reduction of the number of injected antibodies should not affect substantially the treatment efficacy.

SimB16: Modeling Induced Immune System Response against B16-Melanoma

Immunological therapy of progressive tumors requires not only activation and expansion of tumor specific cytotoxic T lymphocytes (CTLs), but also an efficient effector phase including migration of CTLs in the tumor tissue followed by conjugation and killing of target cells. We report the application of an agent-based model to recapitulate both the effect of a specific immunotherapy strategy against B16-melanoma in mice and the tumor progression in a generic tissue section. A comparison of the in silico results with the in vivo experiments shows excellent agreement. We therefore use the model to predict a critical role for CD137 expression on tumor vessel endothelium for successful therapy and other mechanistic aspects. Experimental results are fully compatible with the model predictions. The biologically oriented in silico model derived in this work will be used to predict treatment failure or success in other pre-clinical conditions eventually leading new promising in vivo experiments.

In vitro migration of cytotoxic T lymphocyte derived from a colon carcinoma patient is dependent on CCL2 and CCR2

BackgroundInfiltration of colorectal carcinomas (CRC) with T-cells has been associated with good prognosis. There are some indications that chemokines could be involved in T-cell infiltration of tumors. Selective modulation of chemokine activity at the tumor site could attract immune cells resulting in tumor growth inhibition. In mouse tumor model systems, gene therapy with chemokines or administration of antibody (Ab)-chemokine fusion proteins have provided potent immune mediated tumor rejection which was mediated by infiltrating T cells at the tumor site. To develop such immunotherapeutic strategies for cancer patients, one must identify chemokines and their receptors involved in T-cell migration toward tumor cells.MethodsTo identify chemokine and chemokine receptors involved in T-cell migration toward CRC cells, we have used our previously published three-dimensional organotypic CRC culture system. Organotypic culture was initiated with a layer of fetal fibroblast cells mixed with collagen matrix in a 24 well tissue culture plate. A layer of CRC cells was placed on top of the fibroblast-collagen layer which was followed by a separating layer of fibroblasts in collagen matrix. Anti-CRC specific cytotoxic T lymphocytes (CTLs) mixed with fibroblasts in collagen matrix were placed on top of the separating layer. Excess chemokine ligand (CCL) or Abs to chemokine or chemokine receptor (CCR) were used in migration inhibition assays to identify the chemokine and the receptor involved in CTL migration.ResultsInclusion of excess CCL2 in T-cell layer or Ab to CCL2 in separating layer of collagen fibroblasts blocked the migration of CTLs toward tumor cells and in turn significantly inhibited tumor cell apoptosis. Also, Ab to CCR2 in the separating layer of collagen and fibroblasts blocked the migration of CTLs toward tumor cells and subsequently inhibited tumor cell apoptosis. Expression of CCR2 in four additional CRC patients' lymphocytes isolated from infiltrating tumor tissues suggests their role in migration in other CRC patients.ConclusionsOur data suggest that CCL2 secreted by tumor cells and CCR2 receptors on CTLs are involved in migration of CTLs towards tumor. Gene therapy of tumor cells with CCL2 or CCL2/anti-tumor Ab fusion proteins may attract CTLs that potentially could inhibit tumor growth.

A three-dimensional organotypic assay to measure target cell killing by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) mediate antigen- and cell–cell contact dependent killing of target cells, such as cancer cells and virus-infected cells. In vivo, this process requires the active migration of CTL towards and away from target cells. We here describe an organotypic 3D collagen matrix assay to monitor CTL migration together with CTL-mediated killing of target cells. The assay supports both, time-lapse microscopy of killing dynamics as well as population analysis of killing after matrix digestion and flow cytometry. The assay was used to assess the detrimental effect of cyclosporine A (CsA) present during CTL activation, which caused an inhibition of CTL-target cell conjugation and strongly impaired CTL-mediated killing, particularly at low effector–target ratios. Thus, the organotypic assay is useful to monitor spatiotemporal control mechanisms of cytotoxic immune effector functions.

Role of CD44 in CTL-induced acute liver injury in hepatitis B virus transgenic mice

There are many uncertain points regarding leukocyte movement in the liver, especially interaction between liver sinus endothelial cells (LSECs) and cytotoxic T lymphocytes (CTLs). We examined the role of CD44 in these interactions using the hepatitis model. CTLs were administered to hepatitis B virus transgenic mice (HBVTg) mice and HBVTg x CD44 knockout (KO) mice, and alanine aminotransferase activity (ALT), number of intrahepatic leukocytes, cytokines, and chemokine mRNA level were examined. To determine the number and distribution of CTLs in the liver, 5,6-carboxyfluoroscein succinimidyl ester (CFSE)-labeled CTLs was administered to HBVTg mice with or without CD44. Serum ALT activity increased after 12 h, although it had declined to 4 h in the CD44KO x HBVTg mice after CTL injection. Similarly, the levels of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and macrophage inflammatory protein (MIP)-2 mRNAs were reduced in 4 h, although the levels were increased after 12 h in the CD44KO x HBVTg mice. The number of apoptotic hepatocytes increased intentionally at 24 h in the CD44KO x HBVTg livers, and this was thought to result from the lower activity of initial nuclear factor kappa B (NF-kappaB). Although the number of CTLs was lower at 4 h in the CD44KO x HBVTg livers, the difference of intercellular adhesion molecule (ICAM)-1 and CD86 expression on LSECs was not detected. CD44 exerts and important effect on LSECs for CTL migration into the hepatocytes. However, because the CD44-deficient state exacerbated hepatic injury, attention is necessary for hepatitis treatment as CD44 target therapy.

Preferential involvement of CX chemokine receptor 4 and CX chemokine ligand 12 in T-Cell migration toward melanoma cells

Our previous analysis of the role of chemokines in T lymphocyte trafficking toward human tumor cells revealed the migration of a melanoma patient's cytotoxic T lymphocytes (CTL) toward autologous tumor cells, resulting in tumor cell apoptosis, in an organotypic melanoma culture. CTL migration was mediated by CX chemokine receptor (CXCR) 4 expressed by the CTL and CX chemokine ligand (CXCL) 12 secreted by the tumor cells, as evidenced by blockage of CTL migration by antibodies to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. Here, we present the results of T cell migration in one additional melanoma patient and T cell and tumor cell analyses for CXCR4 and CXCL12 expression, respectively, in 12 additional melanoma patients, indicating the preferential role of CXCR4 and CXCL12 in CTL migration toward melanoma cells. These studies add to the increasing body of evidence suggesting that CXCL12 is a potent chemoattractant for T cells.