Human Cytotoxic T Lymphocytes Research Articles

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

C-Met+ Cytotoxic T Lymphocytes Exhibit Enhanced Cytotoxicity in Mice and Humans In Vitro Tumor Models.

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.

An Immunoproteasome Activator That Increases MHC Class I Antigen Presentation to Enhance Anti-Tumor Immunity

Evasion of immune destruction is a hallmark of cancer that thwarts the efficacy of immunotherapy and reduces patient benefit. The development of immunotherapies has sparked a revolution in oncology by releasing the power of the human immune system, but the identity of novel, actionable antigens exclusively presented on cancer cells remains elusive. Proteasomes are multi-subunit proteolytic complexes that selectively degrade intracellular proteins. Proteasomes play a critical in controlling myriad cellular processes including metabolism, cell cycle progression, signal transduction, protein quality control and differentiation. Importantly, proteasomes are also the source of most antigenic peptides presented to the immune system on major histocompatibility complex (MHC)-class I molecules. In this process, intracellular and viral proteins are degraded in the cytosol to 8 and 9-amino acid peptides, which are then transported into the endoplasmic reticulum, where they associate with MHC-class I molecules and are presented to the cell surface. Immune cells contain a highly specialized, cytokine-inducible proteasome variant, known as the immunoproteasome, in which the three constitutive catalytic subunits are replaced by homologous catalytic subunits. Immunoproteasomes are specially adapted for a role in MHC class I antigen processing and presentation to CD8 + T-cells. Here, we performed a high-through screen (HTS) to detect novel molecular entities that activated immuno-proteasomal catalytic activity. We identified a curated panel of molecules which not only enhanced hydrolysis of the cell-permeable substrate LLVY-R110 but also enhanced the presentation of MHC class I antigens. E.G7-Ova is a mouse lymphoma cell line derived by electroporation of EL4 cells (C57BL/6, H-2b, T lymphoma) with chicken ovalbumin ( OVA) cDNA. Proteasomes degrade OVA to generate the peptide SIINFEKL that is presented by murine MHC-class I H-2Kb molecules. E.G7-Ova cells were pretreated with compound A and co-cultured with the T-cell hybridoma B3Z that had been genetically-engineered to express a SIINFEKL-restricted t-cell receptor (TCR). Pretreatment of E.G7-Ova cells with compound A significantly increased tumor cell lysis by B3Z cells. The treatment of multiple myeloma (MM) cells with compound A also dramatically enhanced the expression of pan-MHC class I antigens. Treatment of MM cells with compound A increased the intracellular level of the proteasome activator PA28α/β, which is expressed by PSME1/2. Compound A treatment also enhanced hydrolysis of the fluorogenic peptide substrate Ac-ANW-AMC, which is selectively cleaved by the immunoproteasome β5-associated catalytic activity. Treatment of MM cells with compound A followed by mass spectrometry (MS) revealed that immunoproteasome activation modulated the relative presentation of individual antigenic peptides bound to MHC class I molecules. Pre-treatment of MM cells with compound A also significantly enhanced the anti-myeloma activity of human cytotoxic T-cells. Taken together, our results demonstrate that immunoproteasome activation has the potential to overcome immune escape and promote host immunity. In addition, our model systems now allows us to identify actionable tumor antigens on patient tumor cells as an indispensable tool for the development of cancer immunotherapies, e.g., T cell receptor (TCR)-transduced T-cells and patient-specific mRNA and peptide vaccines. Finally, immunoproteasome activators have the potential to contribute not only to cytotoxic T-cell mediated tumor lysis but also the treatment of intracellular infections and autoimmune and neurodegenerative proteinopathies characterized by the pathologic accumulation of toxic, proteinaceous aggregates.

Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

RWD80 Gastrointestinal Adverse Events in Lung Cancer Patients Treated with Ipilimumab and Nivolumab Using Real-World Data

Evidence shows that programmed death receptor-1 (PD-1)-blocking and human cytotoxic T-lymphocyte antigen 4 (CTLA-4)-blocking antibodies display apparent benefits in lung cancer therapy. Further, lung cancer patients who receive nivolumab (PD-1 inhibitor) plus ipilimumab (CLTA-4 inhibitor) in combination have higher overall survival rate than those treated with ipilimumab alone. However, adverse events (AE) are still a concern, especially gastrointestinal (GI) AEs. Since GI AE is one of the most common reasons for patients stopping immunotherapy, we utilized real-world data to gain insight in the incidence and severity of GI AEs among lung cancer patients treated with nivolumab and ipilimumab.

The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:β2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

Toxoplasma gondii Tyrosine-Rich Oocyst Wall Protein: A Closer Look through an In Silico Prism.

Toxoplasmosis is a global threat with significant zoonotic concern. The present in silico study was aimed at determination of bioinformatics features and immunogenic epitopes of a tyrosine-rich oocyst wall protein (TrOWP) of Toxoplasma gondii. After retrieving the amino acid sequence from UniProt database, several parameters were predicted including antigenicity, allergenicity, solubility and physico-chemical features, signal peptide, transmembrane domain, and posttranslational modifications. Following secondary and tertiary structure prediction, the 3D model was refined, and immunogenic epitopes were forecasted. It was a 25.57 kDa hydrophilic molecule with 236 residues, a signal peptide, and significant antigenicity scores. Moreover, several linear and conformational B-cell epitopes were present. Also, potential mouse and human cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes were predicted in the sequence. The findings of the present in silico study are promising as they render beneficial characteristics of TrOWP to be included in future vaccination experiments.

Hyperprogression of a mismatch repair-deficient colon cancer in a humanized mouse model following administration of immune checkpoint inhibitor pembrolizumab.

Immunotherapy is an established treatment modality in oncology. However, in addition to primary or acquired therapy resistance with immune checkpoint blockade (ICB), hyperprogressive disease (HPD) or hyperprogression (HP) with acceleration of tumor growth occurs in a subset of patients receiving ICB therapy. A validated and predictive animal model would help investigate HPD/HP to develop new approaches for this challenging clinical entity. Using human cytotoxic T-cell line TALL-104 injected intraperitoneally into immunodeficient NCRU-nude athymic mice bearing mismatch repair-deficient (MMR-d) human colon carcinoma HCT116 p53-null (but not wild-type p53) tumor xenograft, we observed accelerated tumor growth after PD-1 blockade with pembrolizumab administration. There was increased colon tumor cell proliferation as determined by immunohistochemical Ki67 staining of tumor sections. There was no increase in MDM2 or MDM4/MDMX in the p53-null HCT116 cells versus the wild-type p53-expressing isogenic tumor cells, suggesting the effects in this model may be MDM2 or MDM4/MDMX-independent. Human cytokine profiling revealed changes in IFN-γ, TRAIL-R2/TNFRSF10B, TRANCE/TNFSF11/RANK L, CCL2/JE/MCP-1, Chitinase 3-like 1, IL-4 and TNF-α. This represents a novel humanized HPD mouse model with a link to deficiency of the p53 pathway of tumor suppression in the setting of MMR-d. Our novel humanized preclinical TALL-104/p53-null HCT116 mouse model implicates p53-deficiency in an MMR-d tumor as a possible contributor to HPD/HP and may help with evaluating therapeutic strategies in cancer immunotherapy to extend clinical benefits of ICB’s in a broader patient population.

Targeting the Microtubule-Network Rescues CTL Killing Efficiency in Dense 3D Matrices.

Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors.

Cell Softness Prevents Cytolytic T-cell Killing of Tumor-Repopulating Cells.

Biomechanics is a fundamental feature of a cell. However, the manner by which actomysin tension affects tumor immune evasion remains unclear. Here we show that although cytotoxic T lymphocytes (CTL) can effectively destroy stiff differentiated tumor cells, they fail to kill soft tumor-repopulating cells (TRC). TRC softness prevented membrane pore formation caused by CTL-released perforin. Perforin interacting with nonmuscle myosin heavy-chain 9 transmitted forces to less F-actins in soft TRC, thus generating an inadequate contractile force for perforin pore formation. Stiffening TRC allowed perforin the ability to drill through the membrane, leading to CTL-mediated killing of TRC. Importantly, overcoming mechanical softness in human TRC also enhanced TRC cell death caused by human CTL, potentiating a mechanics-based immunotherapeutic strategy. These findings reveal a mechanics-mediated tumor immune evasion, thus potentially providing an alternative approach for tumor immunotherapy. SIGNIFICANCE: Tumor-repopulating cells evade CD8+ cytolytic T-cell killing through a mechanical softness mechanism, underlying the impediment of perforin pore formation at the immune synapse site.

Extracellular Cystatin F Is Internalised by Cytotoxic T Lymphocytes and Decreases Their Cytotoxicity.

Simple SummaryCytotoxic T lymphocytes kill cancer or virally infected cells by exocytosis of lytic granules. This leads to perforin-mediated granzyme entry into the target cell, consequently killing the target cell. Granzymes and perforin are activated by cysteine cathepsins whose activity is regulated by the protein inhibitor cystatin F. Since cystatin F can be secreted by a range of cancer and immune cells in tumour microenvironments, we here investigated whether extracellular cystatin F can be taken up by and affect the function of cytotoxic T lymphocytes. We demonstrated cystatin F uptake into cytotoxic T lymphocytes, down-regulation of target peptidases, and reduced target cell killing. Overall, our results indicate that cystatin F is an important mediator that can impair the killing efficiency of cytotoxic T lymphocytes and thus suggest that it is a possible target for cancer immunotherapy.Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function.

Migration of Cytotoxic T Lymphocytes in 3D Collagen Matrices

CD8+ cytotoxic T lymphocytes (CTL) and natural killer cells are the main cytotoxic killer cells of the human body to eliminate pathogen-infected or tumorigenic cells (also known as target cells). To find their targets, they have to navigate and migrate through complex biological microenvironments, a key component of which is the extracellular matrix (ECM). The mechanisms underlying killer cell’s navigation are not well understood. To mimic an ECM, we use a matrix formed by different collagen concentrations and analyze migration trajectories of primary human CTLs. Different migration patterns are observed and can be grouped into three motility types: slow, fast, and mixed. The dynamics are well described by a two-state persistent random walk model, which allows cells to switch between slow motion with low persistence and fast motion with high persistence. We hypothesize that the slow motility mode describes CTLs creating channels through the collagen matrix by deforming and tearing apart collagen fibers and that the fast motility mode describes CTLs moving within these channels. Experimental evidence supporting this scenario is presented by visualizing migrating T cells following each other on exactly the same track and showing cells moving quickly in channel-like cavities within the surrounding collagen matrix. Consequently, the efficiency of the stochastic search process of CTLs in the ECM should strongly be influenced by a dynamically changing channel network produced by the killer cells themselves.

Defective Interferon Gamma Production by Tumor-Specific CD8+ T Cells Is Associated With 5'Methylcytosine-Guanine Hypermethylation of Interferon Gamma Promoter.

Interferon gamma (IFNγ) supports effector responses of CD8+ cytotoxic T lymphocytes (CTLs) and is a surrogate marker for detection of antigen-specific T cells. Here, we show that tumor-specific CTL clones have impaired IFNγ expression and production upon activation. Assessment of the relationship between IFNγ production and the 5′methylcytosine-guanine (CpG) dinucleotide methylation of the IFNγ promoter using bisulfite treatment has shown that IFNγ− CTL clones accumulates CpG hypermethylation within the promoter at key transcription factor binding sites (−186 and −54), known to be vital for transcription. We confirmed these findings using ex vivo isolated and short-term expanded bulk tumor-specific CTL lines from four cancer patients and demonstrated that IFNγ methylation inversely correlates with transcription, protein level, and cytotoxicity. Altogether, we propose that a sizeable portion of human tumor-specific CTLs are deficient in IFNγ response, contributed by CpG hypermethylation of the IFNγ promoter. Our findings have important implications for immunotherapy strategies and for methods to detect human antigen-specific T cells.

Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of micebearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation.

Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.

Sequential adjustment of cytotoxic T lymphocyte densities improves efficacy in controlling tumor growth

Understanding the human cytotoxic T lymphocyte (CTL) biology is crucial to develop novel strategies aiming at maximizing their lytic capacity against cancer cells. Here we introduce an agent-based model, calibrated on population-scale experimental data that allows quantifying human CTL per capita killing. Our model highlights higher individual CTL killing capacity at lower CTL densities and fits experimental data of human melanoma cell killing. The model allows extending the analysis over prolonged time frames, difficult to investigate experimentally, and reveals that initial high CTL densities hamper efficacy to control melanoma growth. Computational analysis forecasts that sequential addition of fresh CTL cohorts improves tumor growth control. In vivo experimental data, obtained in a mouse melanoma model, confirm this prediction. Taken together, our results unveil the impact that sequential adjustment of cellular densities has on enhancing CTL efficacy over long-term confrontation with tumor cells. In perspective, they can be instrumental to refine CTL-based therapeutic strategies aiming at controlling tumor growth.

A first-in-human, multicenter, open label, phase I study in patients with advanced and/or refractory solid malignancies to evaluate the safety of intravenously administered ATOR-1015.

TPS2653 Background: ATOR-1015 is a human bispecific IgG1 antibody targeting cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and the tumor necrosis factor receptor superfamily member 4, OX40 (also known as CD134). Both in vitro and in vivo, ATOR-1015 induces activation of cytotoxic T cells and depletion of regulatory T cells (1). In syngeneic tumor models, using human OX40 transgenic mice cross-reacting with both targets, ATOR-1015 is demonstrated to localize to the tumor. Further, the effects of ATOR-1015 are shown to occur in the tumor area and not in the spleen (1). Treatment with ATOR-1015 also reduces tumor growth and improves survival in several tumor models in mice, including bladder, colon and pancreatic cancer (1). The non-clinical safety profile and the pharmacokinetics were established in cynomolgus monkeys and the data were used for the dosing schedule. Methods: This is a multicenter, open-label, dose escalation study enrolling patients with advanced and/or refractory solid malignancies (NCT03782467). The primary objective of the study is to determine the maximum tolerated dose (MTD) or the recommended phase 2 dose (RP2D) and to establish the safety profile of ATOR-1015. ATOR-1015 is administered intravenously biweekly as a single agent until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. The study will start with single patient cohorts until grade 2 toxicities are observed, thereafter the study follows a modified 3+3 design. At the MTD/RP2D, or a lower dose, up to 20 patients are planned for additional safety and efficacy evaluation. Study enrollment was initiated in January 2019. A total of up to 53 patients are estimated to be enrolled in the study. (1) Månsson Kvarnhammar et al. Journal for ImmunoTherapy of Cancer 2018; 6(Suppl 1):115. Abstract P683. Clinical trial information: NCT03782467.

Repression of osteopontin by IRF8 regulates a novel immunosuppressive checkpoint

Abstract While current immune checkpoint inhibitors (ICIs) have shown efficacy in a wide variety of human cancers, many patients and cancer types fail to respond to ICI immunotherapy, suggesting the presence of other immunoinhibitory checkpoints. We report here that a deficiency in the transcription factor interferon regulatory factor 8 (IRF8) led to a loss of function of cytotoxic T lymphocytes (CTLs) in both an allograft tumor and a peptide vaccine model. However, a T cell-specific knockout and a mixed chimera model demonstrated that IRF8 suppressed CTL activation through a CTL-extrinsic mechanism. This was found to be through IRF8-dependent suppression of the matricellular protein osteopontin (OPN). During colorectal cancer (CRC) development, IRF8 is downregulated in both tumor-associated CD11b+Ly6CloLy6Ghi myeloid cells and colon epithelial cells, where it represses OPN expression by binding to its promoter. Accordingly, in both mouse CRC models and human CRC patients, transcriptional downregulation of IRF8 was accompanied by an upregulation of OPN at the transcriptional and protein level. Enhanced levels of OPN were found to negatively correlate with patient survival. In vitro stimulation of mouse and human CTLs in the presence of OPN led to decreased activation and interferon gamma secretion. Similarly, deletion of spp1, which encodes for OPN, from murine CT26 colon adenocarcinoma cells caused increased susceptibility to CTL-mediated lysis and enhanced CTL activation. As such, tumor and myeloid-derived OPN may function as a novel immune checkpoint to restrain host CTL activation.

Abstract B165: Investigating in vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model

Abstract Introduction: With the advent of immunotherapy (IT) against various cancers, its applications to other cancers have been extensively investigated. However, it has been a challenge to apply IT to chordomas, due to lack of clinically translatable in vivo models. Currently, there are no well-established murine chordoma cell lines that can be injected to syngeneic mice or no transgenic mouse models that develop chordomas spontaneously, which would allow us to study the interaction between murine chordomas and murine immune cells. Hence, we aimed to develop a humanized mouse model, where human immune cells are engrafted into immunodeficient mice, to study the interaction between human immune system and human chordomas. We also sought to utilize it to investigate synergistic effect between IT and radiation therapy (RT) against chordoma. Materials and Methods: Fifteen 10-12-week-old NSG mice, which lack mouse T-cells, B cells, and NK cells as well as functional mouse macrophages, were sublethally (1.5Gy) irradiated and then implanted with fetal thymic tissue and CD34+ stem cells that had been harvested from a fetus, whose HLA-types were partially-matched with those of the U-CH1 chordoma cell line. Reconstitution of immune cells in NSG mice was confirmed eight weeks post-transplantation, and then each animal (15 humanized NSG mice and 12 naïve NSG mice) was injected with U-CH1 cell suspension bilaterally and subcutaneously. Next, they were treated for 4 weeks as follows: A) control, isotype antibodies (Abs) injection (n=3), B) anti-human-PD-1 Abs (n=4, 10 mg/kg, 3 times/week for 4 weeks), C) RT + isotype Abs (n=3, unilaterally to the left-sided tumor, 8Gy x 4), D) anti-human-PD-1 Abs and RT (n=5), E) naïve NSG mice (n=6, without the engraftment of human immune cells) + isotype, and F) naïve NSG mice (n=6) + anti-human-PD-1 Abs. During and after the treatment, anti-tumor activities were monitored via tumor size, flow cytometry, qRT-PCR, and immunohistochemistry. Results: Eight weeks after stem cell engraftment, human peripheral blood mononuclear cells (PBMCs) of 43.8% among all PBMCs (human + mouse), human T-cells of 23.4% among human PBMCs, human CD8+ T-cells of 24.3% among human T-cells, and other lymphocytes such as B cells, macrophages, and NK cells were observed in peripheral blood of humanized mice via flow cytometry, which confirmed humanization. One week after the treatment, on the irradiated side, (D) demonstrated lowest tumor volume, highest number of human PBMCs, highest % of CD8+ human (cytotoxic) T-cells, highest % of CD45RO+CD4+ human (memory) T-cells, and lowest % of PD-1+CD8+ human (exhausted) T-cells in the tumors via flow cytometry, highest IFN-gamma in the tumors via qRT-PCR, and highest CD8+ human (cytotoxic) T-cells via immunohistochemistry, compared to the other five groups with statistical significance. Of note, on the nonirradiated side, a similar trend was observed with D) harboring the smallest tumor compared to the others (P=0.09), suggesting the abscopal effect. Finally, there were no statistically significant differences amongst (A) humanized NSG mice with isotype-control antibodies, (E) naïve NSG mice with isotype-control antibodies, and (F) naïve NSG mice with anti-PD-1 antibodies on either sides, indicating that HLA-partially-mismatched immune cells derived from the fetus donor were not able to eradicate U-CH1 chordoma cells. Conclusions: We demonstrated that this humanized mouse model could be a revolutionary platform to investigate IT against rare cancers such as chordomas, where murine equivalent cell lines are not available to date, which hinders us from utilizing syngeneic or transgenic mouse models to study IT. The direct synergistic effect between IT and RT against chordoma as well as the potential abscopal effect was observed, evidenced by lowest tumor volume and highest cytotoxic T-cells and memory T-cells. Citation Format: Wataru Ishida, Kyle L. McCormick, Aayushi Mahajan, Eric Feldstein, Michael Lim, Jeffrey N. Bruce, Peter D. Canoll, Sheng-fu L Lo. Investigating in vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B165.

Abstract B114: Cancer immunotherapy combining oral vaccination of recombinant Bifidobacterium longum displaying human Wilms’ tumor 1 protein and anti-PD-1 checkpoint blockade for solid tumors in mice experimental model

Abstract Nowadays, cancer immunotherapies such as immune-checkpoint inhibitors, chimeric antigen receptor T-cells and cancer vaccines have achieved great success in cancer therapy. Nevertheless, increasing cancer antigen-specific immune responses is necessary to obtain significant therapeutic effect. Recently, we had developed an oral cancer vaccine utilizing recombinant Bifidobacterium longum displaying murine Wilms’ tumor 1 antigen (B. longum 420) and had showed significant antitumor effect against WT1-positive leukemic cells by induction of WT1-specifc cytotoxic T-cell (CTL)-mediated immunity via activation of gut mucosal immune system in mice. In this study, we constructed B. longum displaying modified-human WT1 (B. longum 440) and investigated the antitumor effect against solid tumors in mice experimental model. Wild-type B. longum 105-A strain was transformed by electroporation by E. coli-B. longum shuttle vector and recombinant B. longum 440 was constructed. B. longum 440 was displaying human WT1 protein that had modified human WT1-CTL epitope to increase the affinity for MHC-class I. 1×106 of TRAMP-C2, a murine prostate cancer, MBT-2, a murine bladder cancer, and 3LL, a murine lung cancer cell lines were subcutaneously inoculated into mice respectively. All the cell lines were endogenously expressing WT1 protein. After tumor formation, 1×109 of B. longum 440, B. longum 2012 (negative control), or PBS were orally administered 5 times a week for following weeks. PD1-antibody therapy was combined with B. longum 440 therapy to investigate synergistic antitumor effect of immune checkpoint inhibitor against MBT-2. We also performed in vitro co-culture experiments by using B. longum 440, human dendritic cells (DCs) and CTLs derived from human peripheral blood mononuclear cells (PBMCs) to investigate the immune-interaction between B. longum 440 and human immune cells responsible for WT1-spcific immunity. As the results, oral vaccination of B. longum 440 induced significantly higher antitumor effect against TRAMP-C2 and 3LL compared with other therapies, and significantly prolonged overall survival (p<0.05). The combination therapy of B. longum 440 and anti-PD-1 induced substantially higher antitumor effect against MBT-2 tumor than B. longum 440 or PD-1 single therapy, and 40% of the mice completely suppressed the tumor growth. Significantly higher WT1-CTL-epitope, Db126 and mp235-specific IFN-γ and IL-2 production in CD4 and CD8 T-cells were seen in spleen cells isolated from B. longum 440-immunized mice, suggesting that human B. longum 440 could induce WT1-specific T-cell responses in mice as well as murine B. longum 420 as previously reported. Regarding in vitro assay, after co-culturing of B. longum 440 and human DCs, matured DCs that ingested WT1 protein were successfully induced. Then, WT1-specific CTLs were induced by co-culturing of the matured DCs and suspended PBMCs in vitro. As the results, those WT1-specific CTLs elicited significantly higher cell cytotoxicity against A549, a human non-small lung cancer cell line, and T24, a grade 3 human bladder cancer cell line compared to control B. longum 2012 (p<0.05), supporting that B. longum 440 might induce functional WT1-specific CTLs in human. In conclusion, we demonstrated that oral cancer vaccination by using B. longum 440 could induced antitumor effect against prostate cancer, lung cancer and bladder cancer in mice experimental model. In addition, we showed the synergistic antitumor effect of combination therapy of anti-PD-1 and B. longum 440 against bladder cancer. Our findings from in vitro co-culture experiments also supported that combining B. longum 440 cancer vaccines with checkpoint inhibitors has the potential to mediate tumor regression in human. Therefore, B. longum could be a promising candidate of combination immunotherapy with immune checkpoint inhibitors including pembrolizumab, an anti-PD-1 antibody therapy for treatment of advanced bladder cancer. Citation Format: Koichi Kitagawa, Maho Tatsumi, Mako Kato, Shota Komai, Yoshiko Hashii, Takane Katayama, Toshiro Shirakawa. Cancer immunotherapy combining oral vaccination of recombinant Bifidobacterium longum displaying human Wilms’ tumor 1 protein and anti-PD-1 checkpoint blockade for solid tumors in mice experimental model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B114.

Cell Softness Prevents Cytolytic T Cell Killing of Tumor-Repopulating Cells

Biomechanics is a fundamental feature of a cell. The manner by which a mechanical feature could affect immune evasion of tumor cells remains an enigma. Here we show that although cytotoxic T lymphocytes (CTL) can effectively destroy stiff differentiated tumor cells, they fail to kill tumor-repopulating cells (TRC), whose softness effectively impedes the pore formation process through perforin released from CTLs. Mechanistically, perforin interacting with nonmuscle myosin heavy chain 9 transmits forces to fewer F-actins in soft TRCs, thus inducing a weak contractile force which is inadquate for pore formation by perforin. Stiffening soft TRCs confers perforin the drilling ability, leading to CTLs killing the TRCs, thus achieving a better tumor immunotherapeutic outcome. More importantly, overcoming the mechanical softness also enhances the killing of human TRCs by human CTLs. These findings provide mechanics-based immunotherapeutic strategies with potential clinical applications.