Cytotoxicity Of Cytotoxic T Lymphocytes Research Articles

Peroxisome Proliferator-Activated Receptor δ Suppresses the Cytotoxicity of CD8+ T Cells by Inhibiting RelA DNA-Binding Activity.

The molecular mechanisms regulating CD8+ cytotoxic T lymphocytes (CTL) are not fully understood. Here, we show that the peroxisome proliferator-activated receptor δ (PPARδ) suppresses CTL cytotoxicity by inhibiting RelA DNA binding. Treatment of ApcMin/+ mice with the PPARδ agonist GW501516 reduced the activation of normal and tumor-associated intestinal CD8+ T cells and increased intestinal adenoma burden. PPARδ knockout or knockdown in CTLs increased their cytotoxicity against colorectal cancer cells, whereas overexpression of PPARδ or agonist treatment decreased it. Correspondingly, perforin, granzyme B, and IFNγ protein and mRNA levels were higher in PPARδ knockout or knockdown CTLs and lower in PPARδ overexpressing or agonist-treated CTLs. Mechanistically, we found that PPARδ binds to RelA, interfering with RelA-p50 heterodimer formation in the nucleus, thereby inhibiting its DNA binding in CTLs. Thus, PPARδ is a critical regulator of CTL effector function. Significance: Here, we provide the first direct evidence that PPARδ plays a critical role in suppressing the immune response against tumors by downregulating RelA DNA-binding activity. This results in decreased expression of perforin, granzyme B, and IFNγ. Thus, PPARδ may serve as a valuable target for developing future cancer immunotherapies.

DC-derived CXCL10 promotes CTL activation to suppress ovarian cancer

This study investigates the role of dendritic cells (DCs), with a focus on their CXCL10 marker gene, in the activation of cytotoxic T lymphocytes (CTLs) within the ovarian cancer microenvironment and its impact on disease progression. Utilizing scRNA-seq data and immune infiltration analysis, we identified a diminished DC presence in ovarian cancer. Gene analysis pinpointed CXCL10 as a key regulator in OV progression via its influence on DCs and CTLs. Prognostic analysis and in vitro experiments substantiated this role. Our findings reveal that DC-derived CXCL10 significantly affects CTL activation and proliferation. Reduced CXCL10 levels hinder CTL cytotoxicity, promoting ovarian cancer cell migration and invasion. Experimental studies using animal models have provided further evidence that the capacity of CTLs to suppress tumor development is significantly diminished when treated with DCs that have low expression of CXCL10. Dendritic cell-derived CXCL10 emerges as a pivotal factor in restraining ovarian cancer growth and metastasis through the activation of cytotoxic T lymphocytes. This study sheds light on the crucial interplay within the ovarian cancer microenvironment, offering potential therapeutic targets for ovarian cancer treatment.

FOXP4-AS1 promotes CD8+ T cell exhaustion and esophageal cancer immune escape through USP10-stabilized PD-L1.

Esophageal cancer (EC) is the 9th most frequently diagnosed malignancy globally with unfavorable prognosis. Immune escape is one of the principal factors leading to poor survival, however, the mechanism underlying immune escape remains largely uninvestigated. The xenograft mouse model and EC cell-CD8+ cytotoxic T lymphocytes (CTLs) co-culture system were established. Immunohistochemistry, qRT-PCR or western blot were employed to detect the levels of long non-coding RNA (lncRNA) FOXP4-AS1, PD-L1, USP10 and other molecules. The abundance of T cells, cytokine production and cell apoptosis were monitored by flow cytometry. The viability of CTLs was assessed by Trypan blue staining. The binding between FOXP4-AS1 and USP10 was validated by RNA pull-down assay, and the interaction between USP10 and PD-L1, as well as the ubiquitination of PD-L1, were detected by co-immunoprecipitation. The elevation of FOXP4-AS1 in EC was associated with decreased CTL abundance, and upregulated PD-L1 facilitated CTL apoptosis in EC. FOXP4-AS1 accelerated EC tumor growth by decreasing the abundance of tumor infiltrating CTLs in vivo. FOXP4-AS1 inhibited the viability of CTLs and facilitated the cytotoxicity and exhaustion of CTLs. In Kyse 450 cell-CTL co-culture system, FOXP4-AS1 suppressed the viability and abundance of CTLs, and inhibited EC cell apoptosis via PD-L1. Mechanistically, FOXP4-AS1 regulated the ubiquitination of PD-L1 through deubiquitinating enzyme USP10. FOXP4-AS1 promoted CTL exhaustion and EC immune escape through USP10-stabilized PD-L1. HIGHLIGHTS: PD-L1 facilitated CD8+ T cell apoptosis in EC. Upregulated FOXP4-AS1 promoted EC tumor growth by inhibiting the viability and facilitating the cytotoxicity and exhaustion of tumor infiltrating CD8+ T cells. FOXP4-AS1 suppressed the viability and abundance of CD8+ T cells through USP10-mediated deubiquitination of PD-L1.

BCL2A1 neoepitope-elicited cytotoxic T lymphocytes are a promising individualized immunotherapy of pancreatic cancer.

Conventional treatments have shown a limited efficacy for pancreatic cancer, and immunotherapy is an emerging option for treatment of this highly fatal malignancy. Neoantigen is critical to improving the efficacy of tumor-specific immunotherapy. The cancer and peripheral blood specimens from an HLA-A0201-positive pancreatic cancer patient were subjected to next-generation sequencing, and bioinformatics analyses were performed to screen high-affinity and highly stable neoepitopes. The activation of cytotoxic T lymphocytes (CTLs) by dendritic cells (DCs) loaded with mutBCL2A111-20 neoepitope targeting a BCL2A1 mutant epitope was investigated, and the cytotoxicity of mutBCL2A111-20 neoepitope-specific CTLs to pancreatic cancer cells was evaluated. The mutBCL2A111-20 neoepitope was found to present a high immunogenicity and induce CTLs activation and proliferation, and these CTLs were cytotoxic to mutBCL2A111-20 neoepitope-loaded T2 cells and pancreatic cancer PANC-1-Neo and A2-BxPC-3-Neo cells that overexpressed mutBCL2A111-20 neoepitopes, appearing to be a targeting neoepitope specificity. In addition, high BCL2A1 expression correlated with a low 5-yr progression-free interval among pancreatic cancer patients. Our findings provide experimental supports to individualized T cell therapy targeting mutBCL2A111-20 neoepitopes, and provide an option of immunotherapy for pancreatic cancer.

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.

Mathematical modeling identifies LAG3 and HAVCR2 as biomarkers of T cell exhaustion in melanoma

Cytotoxic T lymphocytes (CTLs) control tumors via lysis of antigen-presenting targets or through secretion of cytokines such as interferon-γ (IFNG), which inhibit tumor cell proliferation. Improved understanding of CTL interactions within solid tumors will aid the development of immunotherapeutic strategies against cancer. In this study, we take a systems biology approach to compare the importance of cytolytic versus IFNG-mediated cytostatic effects in a murine melanoma model (B16F10) and to dissect the contribution of immune checkpoints HAVCR2, LAG3, and PDCD1/CD274 to CTL exhaustion. We integrated multimodal data to inform an ordinary differential equation (ODE) model of CTL activities inside the tumor. Our model predicted that CTL cytotoxicity played only a minor role in tumor control relative to the cytostatic effects of IFNG. Furthermore, our analysis revealed that within B16F10 melanomas HAVCR2 and LAG3 better characterize the development of a dysfunctional CTL phenotype than does the PDCD1/CD274 axis.

Altered HLA-A2-restricted TP53 epitope induces specific CTL cytotoxicity against hepatocellular carcinoma.

High-frequency mutation of the TP53 tumor suppressor gene is observed in multiple human cancers, which promotes cancer progression. However, the mutated gene-encoded protein may serve as a tumor antigen to elicit tumor-specific immune responses. In this study, we detected widespread expression of shared TP53-Y220C neoantigen in hepatocellular carcinoma with low affinity and low stability of binding to HLA-A0201 molecules. We substituted the amino acid sequences VVPCEPPEV with VLPCEPPEV in the TP53-Y220C neoantigen to yield a TP53-Y220C (L2) neoantigen. This altered neoantigen was found to increase affinity and stability and induce more cytotoxic T lymphocytes (CTLs), indicating improvements in immunogenicity. In vitro assays showed the cytotoxicity of CTLs stimulated by both TP53-Y220C and TP53-Y220C (L2) neoantigens against multiple HLA-A0201-positive cancer cells expressing TP53-Y220C neoantigens; however, the TP53-Y220C (L2) neoantigen showed higher cytotoxicity than the TP53-Y220C neoantigen against cancer cells. More importantly, in vivo assays demonstrated greater inhibition of hepatocellular carcinoma cell proliferation by TP53-Y220C (L2) neoantigen-specific CTLs relative to TP53-Y220C neoantigen in zebrafish and nonobese diabetic/severe combined immune deficiency mouse models. The results of this study demonstrate enhanced immunogenicity of the shared TP53-Y220C (L2) neoantigen, which has the potential as dendritic cells or peptide vaccines for multiple cancers.

Rapamycin Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction in Immune Thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized with increased risk of bleeding. CD8+ cytotoxic T lymphocytes (CTLs) of ITP patients express high level of lethal molecules, leading to the lysis and apoptosis of their own platelets, which is an important mechanism of platelet destruction in ITP patients. Accumulating evidence have highlighted that the activation of mTOR signaling pathway promotes the differentiation of CD8+ T cells to CTLs, which leads to the up-regulation of cell-mediated immunity. Several studies have showed that rapamycin, a mTOR inhibitor, was effective in the treatment of relapsed/refractory ITP. However, the endogenous mechanism remains to be explored. The activation of mTOR signaling pathway in CD8+ T cells was determined by flow cytometry. We found that the phosphorylation level of p-mTOR, p-Akt, p-4E-BP1 and p-P70S6K in ITP patients were significantly higher than that in healthy controls. Meanwhile, we found that the levels of perforin and granzyme B in CD8+ T cells in ITP patients were significantly higher than that in healthy controls. In the in vitro experiments, CTLs were isolated from PBMCs of ITP patients or healthy controls and cultured with different concentrations of rapamycin (0-1 uM) for 3 days, and we chose an optimal concentration of 20 nM. There was no significant difference in the expression levels of mTOR, Akt, 4E-BP1, and P70S6K after rapamycin treatment, but the phosphorylation level of p-mTOR, p-Akt, p-4E-BP1, p-P70S6K was significantly inhibited by rapamycin treatment. Likewise, the ratios of p-mTOR/mTOR, p-AKT/AKT, p-4E-BP1/4E-BP1, p-P70S6K/P70S6K were also significantly down-regulated. Flow cytometry analysis found that rapamycin significantly inhibited the expression of perforin and granzyme B (Figure 1) in CD8+ T cells of ITP patients. We further established the ITP murine model by transferring splenocytes of C57BL/6 CD61 knockout mice, immunized against platelets from wild-type syngeneic C57BL/6 mice, into severe combined immune deficient (SCID) mice. Rapamycin was injected intraperitoneally daily at a final concentration 2.0 mg/kg. Our data showed that the rapamycin group had significantly higher platelet counts compared with control group. In summary, our findings suggest that the activation of mTOR signaling pathway-related molecules of CD8+ T cells in patients with ITP are abnormal. These data shed new light on the molecular mechanism of rapamycin action by inhibiting CTLs cytotoxicity. This novel mechanism of rapamycin underlies the potential therapeutic strategy for ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Experimental study on the induction of cytotoxic T lymphocyte killing effects and dendritic-cell-based tumor vaccine prepared by high-intensity focused ultrasound.

This study was conducted to explore the high-intensity focused ultrasound (HIFU) prepared antigen-sensitized dendritic cells (DC) and the induction of cytotoxic T lymphocyte (CTL) killing effects by DC and to observe their anti-tumor immunity effects on BALB/c mice. GM-CSF and IL-4 were used to culture the mouse bone marrow-derived DC. HIFU was used to prepare CT-26 tumor cell antigen-sensitive DC vaccines. The capability of T cell proliferation was detected by 3H-TdR, and the CTL cytotoxicity was detected using standard 4h51Cr release assay. The DC-based tumor vaccine prepared using HIFU irradiation was given to normal BALB/c mice. The mice were injected with CT-26 cancer cells subcutaneously seven days later. Further, the occurrence time of the tumor, its weight and volume on the 20th day was observed, and the allergic DC group challenged using repeated-freezing-thawing method alone with the normal saline control group (negative control group) were used to compare group differences. DC in the HIFU group, tumor cell freeze-thawing group, tumor supernatant group, and phosphate buffer solution (PBS) group could induce T cell proliferation in vitro. However, the ability to induce T cell proliferation of DC in the HIFU group and tumor cell freeze-thawing group was significantly higher than those in the tumor supernatant and PBS groups (P < 0. 05). CTL induced in vitro by DC in the HIFU group, and the tumor cell freeze-thawing group had significant cytotoxicity to colon cancer, being significantly different from those in the tumor supernatant and PBS groups (P < 0.05). There was no significant difference between the cytotoxicity of CTL induced in vitro in the HIFU group and the tumor cell freeze-thawing group (P > 0.05). Additionally, significant differences in the occurrence time of the tumor, its weight and volume on the 20th day, and the median survival time of mice among the HIFU group, the repeated-freezing-thawing group, and the negative control group were observed (P < 0.01 or P < 0.05). There was a significant difference between the HIFU and the repeated-freezing-thawing group (P < 0.05). HIFU prepared antigen-sensitized DC could cause substantial proliferation of T cells and CTL with strong anti-tumor effects. The DC-based tumor vaccine prepared using HIFU irradiation affected active immunization on the tumor occurrence in vitro and was better than the DC-based tumor vaccine prepared using the repeated-freezing-thawing method.

Identification of CD8+ T-cell epitope from multiple myeloma-specific antigen AKAP4.

Multiple myeloma (MM) is a malignant plasma cell disorder affecting mainly the elderly population. Revolutionary progress in immunotherapy has been made recently, including monoclonal antibodies and chimeric antigen receptor T cell (CAR-T) therapies; however, the high relapse rate remains problematic. Therefore, combination therapies against different targets would be a reasonable strategy. In this study, we present a new X-chromosome encoded testis-cancer antigen (CTA) AKAP4 as a potential target for MM. AKAP4 is expressed in MM cell lines and MM primary malignant plasma cells. HLA-A*0201-restricted cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) transduced with an adenovirus vector encoding the full-length AKAP4 gene were demonstrated to lyse AKAP4+ myeloma cells. Seven of the 12 candidate epitopes predicated by the BIMAS and SYFPEITH algorithms were able to bind HLA-A*0201 in the T2 binding assay, of which only two peptides were able to induce CTL cytotoxicity in the co-culture of peptide-loaded human mature dendritic cells and the autologous peripheral blood mononuclear cells (PBMCs) from the same HLA-A*0201 donor. The AKAP4 630–638 VLMLIQKLL was identified as the strongest CTL epitope by the human IFN-γ ELISPOT assay. Finally, the VLMLIQKLL-specific CTLs can lyse the HLA-A*0201+AKAP4+ myeloma cell line U266 in vitro, and inhibit tumor growth in the mice bearing U266 tumors in vivo. These results suggest that the VLMLIQKLL epitope could be used to develop cancer vaccine or T-cell receptor transgenic T cells (TCR-T) to kill myeloma cells.

Blockade of TIM-3 and PD-1 enhances the antitumor effects of MAGE-A11 antigen-specific cytotoxic T lymphocytes

Cancer immunotherapy is an attractive approach for antigen-specific T cell-mediated antitumor therapy, especially for the induction of cytotoxic T lymphocytes (CTLs). An human leukocyte antigen (HLA)-A2-restricted melanoma-associated antigen-A11 (MAGE-A11) peptide was developed that exhibited a potent capacity to induce cytotoxicity towards MAGE-A11-positive breast cancer cells by activating CTLs. However, this antitumor immune response can be suppressed by inhibitory pathways. Programmed death-1 (PD-1) and T cell immunoglobulin domain and mucin domain 3 (TIM-3) pathways are two important pathways involved in the tumor-mediated immune suppression. The present study aimed to augment the efficacy of MAGE-A11 antigen-specific CTLs via blocking PD-1 and TIM-3. The results showed that the expression levels of PD-1 and TIM-3 were unregulated during T cell induction, expansion and target cell killing. Blockade of PD-1 and TIM-3 modulated T cell proliferation, transformation and survival. In addition, treatment of cells with antibodies against PD-1 and TIM-3 enhanced the cytotoxicity of MAGE-A11 antigen-specific CTLs against breast cancer cells. The aforementioned findings suggested that MAGE-A11 antigen-specific CTLs accompanied by PD-1 and TIM-3 blockade could be considered as a potential therapy approach for breast cancer.

Resistance to Anti-PD-1 Therapy is mediated via the Microbiota-Th17-Prostoglandin E2 Axis in the LSLKrasG12D Lung Cancer Model

Abstract We previously demonstrated that anti-PD-1 antibody mediated activation of type 17 T-cells undermined checkpoint inhibitor therapy in the LSLKrasG12D murine lung cancer model. In this study we confirm that the Th17 subset is the primary driver of resistance to anti-PD-1; demonstrate that the ontogeny of dysplasia-associated Th17 cells is driven by microbiota-conditioned macrophages; and identify IL-17-COX-2/Prostoglandin E2 (PGE2) axis as the mediator of CD8+ cytotoxic T-lymphocyte (CTL) de-sensitization to checkpoint inhibitor therapy. Specifically, anti-PD-1 treatment of CD4+ T-cell RORc-deficient LSLKrasG12D mice resulted in a 50% increase in CTL cytotoxicity and a 2.2-fold reduction in tumor burden confirming the critical role of Th17 cells in resistance to therapy. Lung-specific depletion of microbiota reduced Th17 cell prevalence and tumor burden by ~2 and ~1.5 fold, respectively, establishing a link between microbiota and the Th17-driven tumorigenesis. Importantly, interstitial macrophages from microbiota sufficient, but not from microbiota-deficient, mice polarized naïve CD4+ T-cells towards a Th17 phenotype, highlighting their role in bridging microbiota and lung Th17 immunity. Neutralization of IL-17 diminished COX-2/PGE2 levels, whereas inhibition of COX-2 rescued CTL activity and restored tumor suppression in anti-PD-1 antibody-treated mice, revealing the molecular basis of IL-17-driven anti-PD-1 resistance. The clinical implications of these findings are discussed. Supported by grant from DOD grant (to N.E., OGMB190737) and in part through the NIH/NIGMS P20-GM135004 (1P20GM135005-01)

Severely impaired CTL killing is a feature of the neurological disorder Niemann-Pick disease type C1

Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder resulting from mutations in an endolysosomal cholesterol transporter, NPC1. Despite typically presenting with pronounced neurological manifestations, NP-C1 also resembles long-term congenital immunodeficiencies that arise from impairment of cytotoxic T lymphocyte (CTL) effector function. CTLs kill their targets through exocytosis of the contents of lysosome-like secretory cytotoxic granules (CGs) that store and ultimately release the essential pore-forming protein perforin and proapoptotic serine proteases, granzymes, into the synapse formed between the CTL and target cell. We discovered that NPC1 deficiency increases CG lipid burden, impairs autophagic flux through stalled trafficking of the transcription factor EB (TFEB), and dramatically reduces CTL cytotoxicity. Using a variety of immunological and cell biological techniques, we found that the cytotoxic defect arises specifically from impaired perforin pore formation. We demonstrated defects of CTL function of varying severity in patients with NP-C1, with the greatest losses of function associated with the most florid and/or earliest disease presentations. Remarkably, perforin function and CTL cytotoxicity were restored in vitro by promoting lipid clearance with therapeutic 2-hydroxypropyl-β-cyclodextrin; however, restoration of autophagy through TFEB overexpression was ineffective. Overall, our study revealed that NPC1 deficiency has a deleterious impact on CTL (but not natural killer cell) cytotoxicity that, in the long term, may predispose patients with NP-C1 to atypical infections and impaired immune surveillance more generally.

BET inhibitor JQ1 enhances anti-tumor immunity and synergizes with PD-1 blockade in CRC.

Most colorectal cancer (CRC) patients are insensitive to immune checkpoint inhibitors (ICIs) due to the immunosuppressive tumor microenvironment (TME). Epigenetic factors such as the bromo-and extraterminal domain (BET) family proteins may be responsible for the immunosuppressive microenvironment. Previous studies have shown that inhibitors of BET family proteins have the potential to remodel the immunosuppressive TME. However, data on the role of BET inhibitors in immune microenvironment in CRC remains unclear. Here, we evaluated the immunoregulatory role of JQ1, a BET inhibitor, in CRC. Transcriptome sequencing data showed that JQ1 decreased CD274 expression and increased H2Kb expression in MC38 cells. Flow cytometry assays demonstrated that JQ1 decreased cell-surface PD-L1 expression in MC38 and HCT116 cells. Moreover, JQ1 significantly increased cell-surface expression of major histocompatibility complex class I (MHC-I) in MC38 cells and HCT116 cells. Antigen-specific cytotoxic T lymphocytes (CTLs) assay demonstrated that JQ1 enhanced the MHC-I-mediated cytotoxicity of CTLs. Mouse colon cancer cell line MC38 was used to establish the syngeneic mouse tumor model. Compared with the control, JQ1 significantly inhibited tumor growth and prolonged the overall survival of the mice. Besides, JQ1 did not only inhibit tumor growth by enhancing anti-tumor immunity, but also promoted the anti-tumor effect of PD-1 antibody. In addition, our data showed that JQ1 reduced infiltration of intratumoral regulatory T cells (Treg), thus remodeling the immunosuppressive TME. Taken together, these results highlight a new approach that enhances anti-PD-1 sensitivity in CRC.

MicroRNA-206 enhances antitumor immunity by disrupting the communication between malignant hepatocytes and regulatory T cells in c-Myc mice.

Intertumoral accumulation of regulatory T cells (Tregs) has been implicated in the pathogenesis of HCC. Because of poor understanding of the immunosuppression mechanism(s) in HCC, immunotherapy is largely unsuccessful for the treatment of HCC. Hydrodynamic injection (HDI) of c-Myc into mice resulted in enlarged spleens and lethal HCC associated with an increase in hepatic Tregs and depletion of CTLs (cytotoxic T lymphocytes). Malignant hepatocytes in c-Myc mice overproduced TGFβ1, which enhanced the suppressor function of Tregs and impaired the proliferation and cytotoxicity of CTLs. In addition to activating TGFβ signaling, c-Myc synergized with Yin Yang 1 to impair microRNA-206 (miR-206) biogenesis. HDI of miR-206 fully prevented HCC and the associated enlargement of the spleen, whereas 100% of control mice died from HCC within 5-9 weeks postinjection. Mechanistically, by directly targeting errant kirsten ras oncogene (KRAS) signaling, miR-206 impeded the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)axis that drives expression of Tgfb1. By blocking the KRAS/MEK/ERK axis, miR-206 prevented TGFβ1 overproduction, thereby impairing the suppressor function and expansion of Tregs, but enhancing the expansion and cytotoxic program of CTLs. Disrupting the interaction between miR-206 and Kras offset the roles of miR-206 in inhibiting immunosuppression and HCC. Depletion of CD8+ T cells impaired the ability of miR-206 to inhibit HCC. c-Myc-educated hepatocytes promoted immunosuppression by overproducing TGFβ1, which promoted HCC development. miR-206, by attenuating TGFβ1 overproduction, disrupted the communication of malignant hepatocytes with CTLs and Tregs, which prevented HCC. miR-206 represents a potential immunotherapeutic agent against HCC.

Low-Dose Decitabine Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction via Modulating PD-1 Methylation in Immune Thrombocytopenia.

Cytotoxic T lymphocytes (CTLs)-mediated platelet destruction plays an important role in the pathogenesis of primary immune thrombocytopenia (ITP). The programmed cell death protein 1 (PD-1) signaling can turn off autoreactive T cells and induce peripheral tolerance. Herein, we found that the expression of PD-1 and its ligand PD-L1 on CD8+ T cells from ITP patients was decreased. Activating PD-1 pathway by PD-L1-Fc fusion protein inhibited CTLs-mediated platelet destruction in ITP in vitro. PD-1 promoter hypermethylation in CD8+ T cells was found in ITP patients, resulting in decreased PD-1 expression. The demethylating agent decitabine at a low dose was proved to restore the methylation level and expression of PD-1 on CD8+ T cells and reduce the cytotoxicity of CTLs of ITP patients. The phosphorylation levels of phosphatidylinositol 3-kinase (PI3K) and AKT in CD8+ T cells were significantly downregulated by low-dose decitabine. Furthermore, blocking PD-1 could counteract the effect of low-dose decitabine on CTLs from ITP patients. Therefore, our data suggest that the aberrant PD-1/PD-L1 pathway is involved in the pathophysiology of ITP and enhancing PD-1/PD-L1 signaling is a promising therapeutic approach for ITP management. Our results reveal the immunomodulatory mechanism of low-dose decitabine in ITP by inhibiting CTLs cytotoxicity to autologous platelets through PD-1 pathway.

CNOT4 suppresses non-small cell lung cancer progression and is required for effector cytolytic T lymphocytes cell responses to lung cancer cells

The therapeutic options of non-small cell lung cancer (NSCLC) are limited, although a combination of targeted therapy and immunotherapy is promising. To explore novel targets for immunotherapy, we explored the role of Ccr4-Not transcription complex subunit 4 (CNOT4) in NSCLC. The expression of CNOT4 in tumor tissues was determined by immunohistochemistry staining and western blotting. The cell lines that stably express CNOT4 were established in H1299 and A549 cells. Direct cell counting, MTT assay, and colony formation were used to determine the ability of cell proliferation. Cell apoptosis and cell cycle were next analyzed by PI/Annexin V staining. Cell invasion and migration were examined by transwell assays. To further explore the function of CNOT4 in cytotoxic T lymphocytes (CTLs) mediated cytotoxicity, an in vitro co-culture system of CNOT4 overexpressing and control H1299 cells with CTLs was developed. CNOT4 was down-regulated in tumor tissues compared with paired normal tissues from patients with lung cancers. CNOT4 overexpression significantly inhibited tumor cell proliferation, colony formation, cell migration, and invasion, but promoted cell apoptosis. Furthermore, overexpression of CNOT4 enhanced cytotoxicity of CTLs to H1299. CNOT4 functions as a potential tumor suppressor of NSCLC via inhibiting tumor cell function and increasing the sensitivity to CTLs.

Morphine-3-glucuronide upregulates PD-L1 expression via TLR4 and promotes the immune escape of non-small cell lung cancer.

Objective:Patients with cancer pain are highly dependent on morphine analgesia, but studies have shown a negative correlation between morphine demand and patient outcomes. The long-term use of morphine may result in abnormally elevated serum morphine-3-glucuronide (M3G) levels. Hence, the effects of M3G on tumor progression are worth studying.Methods:The effects of M3G on PD-L1 expressions in human non-small cell lung cancer (NSCLC) cell lines were first evaluated. Activation of TLR4 downstream pathways after M3G treatment was then determined by Western blot. The effects of M3G on human cytotoxic T lymphocytes (CTL) cytotoxicity and INF-γ release was also detected. Finally, the LLC murine lung adenocarcinoma cell line were used to establish a murine lung cancer model, and the effects of M3G on tumor growth and metastasis were determined.Results:M3G promoted the expressions of PD-L1 in the A549 and H1299 cell lines in a TLR4-dependent manner (P < 0.05). M3G activated the PI3K and the NFκB signaling pathways, and this effect was antagonized by a TLR4 pathway inhibitor. A PI3K pathway inhibitor reversed the M3G-mediated PD-L1 upregulation. M3G inhibited the cytotoxicity of CTL on A549 cells and decreased the level of INF-γ. Repeated M3G intraperitoneal injections promoted LLC tumor growth and lung metastasis through the upregulation of tumor expressed PD-L1 and the reduction of CTL in the tumor microenvironment.Conclusions:M3G specifically activated TLR4 in NSCLC cells and upregulated PD-L1 expression through the PI3K signaling pathway, thereby inhibiting CTL cytotoxicity and finally promoting tumor immune escape.

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.