T-cell Apoptosis Research Articles

Inactivation of Proprotein Convertases in T Cells Inhibits PD-1 Expression and Creates a Favorable Immune Microenvironment in Colorectal Cancer.

Proprotein convertases (PC) activate precursor proteins that play crucial roles in various cancers. In this study, we investigated whether PC enzyme activity is required for expression of the checkpoint protein programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTL) in colon cancer. Although altered expression of the PC secretory pathway was observed in human colon cancers, only furin showed highly diffuse expression throughout the tumors. Inhibition of PCs in T cells using the general protein-based inhibitor α1-PDX or the pharmacologic inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethylketone repressed PD-1 and exhausted CTLs via induction of T-cell proliferation and apoptosis inhibition, which improved CTL efficacy against microsatellite instable and microsatellite stable colon cancer cells. In vivo, inhibition of PCs enhanced CTL infiltration in colorectal tumors and increased tumor clearance in syngeneic mice compared with immunodeficient mice. Inhibition of PCs repressed PD-1 expression by blocking proteolytic maturation of the Notch precursor, inhibiting calcium/NFAT and NF-κB signaling, and enhancing ERK activation. These findings define a key role for PCs in regulating PD-1 expression and suggest targeting PCs as an adjunct approach to colorectal tumor immunotherapy. SIGNIFICANCE: Protein convertase enzymatic activity is required for PD-1 expression on T cells, and inhibition of protein convertase improves T-cell targeting of microsatellite instable and stable colorectal cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/5008/F1.large.jpg.

Tyrosine kinase inhibition to improve anthracycline-based chemotherapy efficacy in T-cell lymphoma

BackgroundAnthracycline-containing regimens, namely cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) or CHOEP (CHOP + etoposide), represent the current standard of care for patients with newly diagnosed peripheral T-cell lymphomas (PTCLs), although responses are unsatisfactory. In this study, we investigated the pathways able to mitigate the sensitivity to CHOP-based regimens in preclinical models of T-cell lymphoma (TCL) to select agents for the development of CHOP-based drug combinations.MethodsWe performed gene expression profiling of malignant T-cell lines exposed to CHOEP; flow cytometry was employed to study the effects of drug combinations on cell viability, cell cycle distribution, apoptosis and mitochondrial membrane depolarisation. Western blot analyses were performed to study cell signalling downstream of the T-cell receptor and apoptosis. The in vivo effect of the drug combination was tested in xenograft models.ResultsWe highlighted a modulation of tyrosine kinases belonging to the T-cell receptor pathway upon chemotherapy that provided the rationale for combining the tyrosine kinase inhibitor dasatinib with CHOEP. Dasatinib improves CHOEP activity and reduces viability in vitro. Furthermore, combination treatment results in tumour growth inhibition in in vivo xenograft mouse models.ConclusionsOur data provide the rationale for clinical testing of the dasatinib–CHOEP combination in patients with T-cell lymphoma.

Proliferation and apoptosis of experimental animal’s lymphocytes after multiple transplantation of immune cells from opposite behavioral types of donors carried out in the juvenile period

The aim of the study was to determine subpopulation content, proliferative activity and T-lymphocyte apoptosis level in adult mice-recipients that in the juvenile period underwent multiple transplantation of immune cells with different functional properties from opposite behavioral types of donors. Materials and methods. The study was carried out on male mice (CBAxC57BL/6) F1 (n = 190), which underwent transplantation of immune cells with definite functional properties three times starting from the age of 4–5-weeks. Phenotyping of recipients’ spleen cells was carried out by flow cytometry with monoclonal antibodies against CD3+, СD4+, CD8+. Splenocyte proliferation and apoptosis were estimated. Results. Animals that underwent threefold transplantation of immune cells from singeing opposite behavioral types of donors in the juvenile period revealed different functional properties of spleen lymphocytes in adults. The most pronounced changes were detected in the animals receiving the immune cells from donors with passive behavior type. The lymphocytes of these recipients were characterized by relatively low proliferative activity and T-mytogen sensitivity, decreased CD4+, CD8+ apoptosis level under deficient medium conditions and increased level of activation and dexamethasone-induced apoptosis in CD4+ lymphocytes.

Label-free purification of viable human T-lymphocyte cells from a mixture of viable and non-viable cells after transfection by electroporation

Recent developments in chimeric antigen receptor (CAR) T-cell therapy for cancer have shown promising results. CAR T-cells are engineered mainly by AC or DC electroporation to express CAR molecules on their surfaces. Electroporation has detrimental effects on the majority of T-cells and causes cell death via apoptosis or necrosis. These non-viable cells have potentially harmful effects on other healthy T-cells. Because there are no unique biomarkers that pinpoint the apoptosis and necrosis of T-cells, currently available cell separation methods are incapable of purifying healthy T-cells after electroporation. To address this critical issue, we have used dielectrophoretic cell isolation in a simple microfluidics chip. Our results indicate that it is possible to purify cell samples after electroporation and achieve ~100% purity and >90% target cell recovery. Therefore, this work presents a viable solution to a critical need in CAR T-cell manufacturing.

MiRNA-148a-3p Regulates Immunosuppression in DNA Mismatch Repair-Deficient Colorectal Cancer by Targeting PD-L1.

Immunotherapy against the interaction between programmed cell death 1/programmed cell death ligand 1 (PD-L1) has emerged as a promising strategy for colorectal cancer with mismatch repair deficiency (dMMR) or microsatellite instability-high (MSI-H). The study aimed to identify miRNAs that posttranscriptionally control PD-L1 expression on tumor cells and also regulate immune evasion. A comprehensive miRNA screening using The Cancer Genome Atlas (TCGA) dataset (n = 260) combined with eight different miRNA target prediction programs resulted in the identification of a tumor suppressive miRNA, miR-148a-3p, as a potential negative regulator of PD-L1 expression, particularly in dMMR/MSI-H colorectal cancer. Using multiple cohorts of colorectal cancer, including TCGA data, a microarray dataset (n = 148), and formalin-fixed, paraffin-embedded samples (n = 395), we found that the expression of miR-148a-3p was decreased in dMMR/MSI-H tumors, correlating inversely with PD-L1 levels. We demonstrate that miR-148a-3p directly binds to the 3'-untranslated region of PD-L1, thereby reducing whole-cell and cell surface PD-L1 levels in HCT116 and SW837 cell lines. Overexpression of miR-148a-3p repressed IFNγ-induced PD-L1 expression on tumor cells and consequently diminished T-cell apoptosis in a coculture model of IL2-activated T cells and IFNγ-treated tumor cells. In conclusion, our data support a regulatory mechanism of PD-L1 expression on tumor cells and immune suppression via miR-148a-3p downregulation in colorectal cancer. IMPLICATIONS: This study provides novel evidence that miR-148a-3p negatively regulates tumor cell PD-L1 expression and decreased levels of miR-148a-3p contributes to the immunosuppressive tumor microenvironment.

Induction of lymphocyte apoptosis in healthy individuals and patients with rheumatoid arthritis under “cellular neighborhood” in vitro

The aim of the study was to investigate the features of T-lymphocyte apoptosis induced by components of autologous apoptotic cultures in vitro in norm and rheumatoid arthritis in the context of «cellular neighborhood».Materials and methods. Subjects of the study were blood samples of patients with rheumatoid arthritis (RA) and healthy women of comparable age. Developed protocol allowed to differentially evaluate the parameters of proliferation, early and late stages of apoptosis in the «primary» (CFSE-) and «secondary» (CFSE+) induced apoptotic T-lymphocyte cultures. It was estimated the effect of cellular and humoral components of unstimulated, anti-CD3- and dexamethasone-stimulated cells under the conditions of overcrowding and depleted culture media on autologous lymphocytes, cultured under physiological conditions, in norm and RA.Results. Comparative qualitative analysis revealed the features of the processes of T-lymphocyte apoptosis in norm and pathology. Also, the parameters of early and late stages of apoptosis of a «primary» induced culture and «secondary» induced cells after transferring the cellular and humoral components of apoptotic cultures did not differ significantly either initially or during culturing in both investigated groups. But it was a significant increase in the amount of living T-cells in «primary»-induced unstimulated and dexamethasone-stimulated RA patients’ cultures compared to similar donors’ cultures.Conclusion. There was no difference between stimulated with anti-CD3 antibodies cells and the «secondary» induced cultures. Taking into account the absence of significant differences in the parameters of activation apoptosis, the increased number of living cells in RA patients’ cultures relative to donors’ is evidence of contribution of non-autonomous apoptosis effects to cellular homeostasis in RA.

Expression and biological functions of the CCL5-CCR5 axis in oral lichen planus.

Oral lichen planus (OLP) is a chronic inflammatory oral mucosal disorder with T cell-mediated immunological pathogenesis. C-C motif chemokine ligand 5 (CCL5) and its receptor C-C motif chemokine receptor 5 (CCR5) play important roles in the activation and recruitment of T cells. This study sought to explore the expression and biological functions of the CCL5-CCR5 axis in OLP. We examined the expression of the CCL5-CCR5 axis in the peripheral blood and oral tissue of healthy controls and patients with OLP using quantitative real-time PCR, Simple Western assays, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. In addition, we investigated the effects of the CCL5-CCR5 axis on the proliferation, apoptosis and migration of OLP T cells using Cell Counting Kit-8 (CCK8) assay, flow cytometry and transwell assay. We found that the expression of the CCL5-CCR5 axis was significantly elevated both in the peripheral blood and oral tissue of patients with OLP compared with healthy controls. CCL5 not only promoted OLP T-cell proliferation and migration but also inhibited OLP T-cell apoptosis. Moreover, CCR5 inhibition suppressed OLP T-cell proliferation and migration, whereas OLP T-cell apoptosis was promoted. In conclusion, our data suggest that the CCL5-CCR5 axis may be closely related to the inflammatory infiltration of T cells in OLP.

Selective effects of ginseng pectins on galectin-3-mediated T cell activation and apoptosis

Galectin-3 (Gal-3) can induce T-cell activation and apoptosis and plays a role in tumor immune tolerance. Here, we demonstrate that ginseng pectins selectively inhibit Gal-3-induced T-cell apoptosis, while not affecting T-cell activation. This finding stands in contrast to that from the use of modified citrus pectin (MCP) and potato galactan (P-galactan) that inhibit both. Whereas PKC/ERK and ROS/ERK pathways are involved in both T-cell activation and apoptosis, the Ras/PI3K/Akt pathway is unique to T-cell activation. Ginseng pectins selectively inhibit the ROS/ERK pathway. Using the Sarcomar-180 mouse model in which Gal-3 expression is increased, we found that ginseng pectins (but not MCP or P-galactan) significantly promote T-cell proliferation and IL-2 expression, and inhibit tumor growth by 45%. These in vivo data correlate well with selective effects of pectins on Gal-3-mediated T-cell apoptosis and activation. Our study suggests a novel approach for the development of polysaccharide-based agents that target Gal-3 function.

ZBTB24 regulates the apoptosis of human T cells via CDCA7/TRAIL-receptor axis

Mutations in ZBTB24 and CDCA7 cause the Immunodeficiency, Centromeric Instability and Facial Anomalies syndrome type 2 and 3 (ICF2/3), respectively. Most ICF2 patients carry ZBTB24 nonsense mutations and are thus ZBTB24-deficient. Although the immune deficiency in ICF2 patients is primarily regarded as a B-cell defect due to the greatly reduced serum antibodies and circulating memory B cells, the reduced expansions of PBMCs stimulated by mitogens or recall antigens suggest a T-cell defect in these patients as well. However, the molecular mechanisms behind this T-cell dysfunction remain unknown. In the present study, we demonstrated that ZBTB24-deficiency significantly represses the proliferation of human T cells by promoting TRAIL-induced cell death. Downregulation of ZBTB24 in both Jurkat and human primary T cells upregulates the expression of TRAIL and/or its death receptors (TRAIL-R1/2), and induces significant amount of cells to undergo apoptosis. The profound survival defects of ZBTB24-deficient cells are largely reversed by blocking TRAIL/TRAIL-R interactions with exogenous recombinant TRAIL-R2. Moreover, ZBTB24-downregulation reduces the expression of CDCA7, and knockdown of the latter in human T cells results in a phenotype resembling that caused by ZBTB24-depletion. Functionally, overexpression of CDCA7 abrogates the increased apoptosis in ZBTB24-depleted Jurkat T cells. Together, these data indicated that ZBTB24 regulates human T-cell apoptosis via CDCA7/TRAIL-R axis. Our study thus not only provides a molecular explanation for the T-cell defects in ZBTB24-deficient ICF2 patients, but also highlights a convergence between ZBTB24 and CDCA7, the two ICF genes, in modulating the functions of T cells.

Endoplasmic Reticulum Stress Causes Liver Cancer Cells to Release Exosomal miR-23a-3p and Up-regulate Programmed Death Ligand 1 Expression in Macrophages.

Endoplasmic reticulum (ER) stress promotes tumor cell escape from immunosurveillance. However, the underlying mechanisms remain unknown. We hypothesized that ER stress induces hepatocellular carcinoma (HCC) cells to release exosomes, which attenuate antitumor immunity by modulating the expression of programmed death ligand 1 (PD-L1) in macrophages. In this study, we demonstrated that expression of several ER stress markers (glucose-regulated protein 78, activating transcription factor 6, protein kinase R-like ER kinase, and inositol-requiring enzyme 1α) was up-regulated in HCC tissues and negatively correlated with the overall survival and clinicopathological scores in patients with HCC. Expression of ER stress-related proteins positively correlated with CD68+ macrophage recruitment and PD-L1 expression in HCC tissues. High-throughput sequencing analysis identified miR-23a-3p as one of the most abundant microRNAs in exosomes derived from tunicamycin (TM)-treated HCC cells (Exo-TMs). miR-23a-3p levels in HCC tissues negatively correlated with overall survival. Treatment with Exo-TMs up-regulated the expression of PD-L1 in macrophages in vitro and in vivo. Bioinformatics analysis suggests that miR-23a-3p regulates PD-L1 expression through the phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway. This notion was confirmed by in vitro transfection and coculture experiments, which revealed that miR-23a-3p inhibited PTEN expression and subsequently elevated phosphorylated AKT and PD-L1 expression in macrophages. Finally, coculture of T cells with Exo-TM-stimulated macrophages decreased CD8+ T-cell ratio and interleukin-2 production but increased T-cell apoptosis in vitro. Conclusion: ER-stressed HCC cells release exosomes to up-regulate PD-L1 expression in macrophages, which subsequently inhibits T-cell function through an exosome miR-23a-PTEN-AKT pathway. Our findings provide insight into the mechanism how tumor cells escape from antitumor immunity.

Blocking Triggering Receptor Expressed on Myeloid Cells-1-Positive Tumor-Associated Macrophages Induced by Hypoxia Reverses Immunosuppression and Anti-Programmed Cell Death Ligand 1 Resistance in Liver Cancer.

Tumor‐associated macrophages (TAMs) are recognized as antitumor suppressors, but how TAMs behave in the hypoxic environment of hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrated that hypoxia inducible factor 1α induced increased expression of triggering receptor expressed on myeloid cells‐1 (TREM‐1) in TAMs, resulting in immunosuppression. Specifically, TREM‐1‐positive (TREM‐1+) TAMs abundant at advanced stages of HCC progression indirectly impaired the cytotoxic functions of CD8+ T cells and induced CD8+ T‐cells apoptosis. Biological and functional assays showed that TREM‐1+ TAMs had higher expression of programmed cell death ligand 1 (PD‐L1) under hypoxic environment. However, TREM‐1+ TAMs could abrogate spontaneous and PD‐L1‐blockade‐mediated antitumor effects in vivo, suggesting that TREM‐1+ TAM‐induced immunosuppression was dependent on a pathway separate from PD‐L1/programmed cell death 1 axis. Moreover, TREM‐1+ TAM‐associated regulatory T cells (Tregs) were crucial for HCC resistance to anti‐PD‐L1 therapy. Mechanistically, TREM‐1+ TAMs elevated chemokine (C‐C motif) ligand 20 expression through the extracellular signal‐regulated kinase/NF‐κβ pathway in response to hypoxia and tumor metabolites leading to CCR6+Foxp3+ Treg accumulation. Blocking the TREM‐1 pathway could significantly inhibit tumor progression, reduce CCR6+Foxp3+ Treg recruitment, and improve the therapeutic efficacy of PD‐L1 blockade. Thus, these data demonstrated that CCR6+Foxp3+ Treg recruitment was crucial for TREM‐1+ TAM‐mediated anti‐PD‐L1 resistance and immunosuppression in hypoxic tumor environment. Conclusion: This study highlighted that the hypoxic environment initiated the onset of tumor immunosuppression through TREM‐1+ TAMs attracting CCR6+Foxp3+ Tregs, and TREM‐1+ TAMs endowed HCC with anti‐PD‐L1 therapy resistance.

SAT-436-Inhibition of autophagy prolongs recipient survival through accelerating CD8+ T-cell apoptosis in a rat liver transplantation model

SAT-436-Inhibition of autophagy prolongs recipient survival through accelerating CD8+ T-cell apoptosis in a rat liver transplantation model

Induction of apoptosis in CD4(+) T-cells is linked with optimal treatment response in patients with relapsing-remitting multiple sclerosis treated with Glatiramer acetate

BackgroundInduction of T-cell apoptosis constitutes a mechanism of action for Glatiramer Acetate (GA). We investigated whether activation of apoptotic T-cell death may be indicative of optimal treatment response in patients with relapsing-remitting Multiple Sclerosis (RRMS), with respect to radiological activity. MethodsWe studied apoptotic markers on blood T-cells of forty patients with RRMS, 19 patients under GA and 21 patients under interferon-β (IFNβ), by flow cytometry. Patients were relapse-free and were further classified into optimal and sub-optimal responders based on radiological activity. Eighteen patients (8 patients under GA and 10 patients under IFNβ were additionally evaluated at a 12-month follow-up and were relapse- and radiological activity-free. For these patients, apoptosis was also evaluated by molecular techniques. ResultsAt inclusion, optimal responders to GA exhibited increased (23.6 ± 1.976) relative % frequency of CD4(+)AnnexinV(+)7AAD(−) T-cells, compared to sub-optimal responders (14.478 ± 1.204, p = 0.001). Similarly, relative % frequency of caspase-3(+) T-cells was 1.517 ± 0.436 versus 0.45 ± 0.149 (p = 0.041), respectively. Anti-apoptotic molecule bcl-2 showed an inverse pattern 4.532 ± 1.321 versus 13.094 ± 3.987, p = 0.044, respectively. These differences were not evident for IFNβ-treated patients. ConclusionsT-cell apoptotic markers may be applied as a biomarker useful in evaluating optimal treatment response under GA, thus allowing for personalized treatment decisions.

Daclizumab: Mechanisms of Action, Therapeutic Efficacy, Adverse Events and Its Uncovering the Potential Role of Innate Immune System Recruitment as a Treatment Strategy for Relapsing Multiple Sclerosis.

Daclizumab (DAC) is a humanized, monoclonal antibody that blocks CD25, a critical element of the high-affinity interleukin-2 receptor (IL-2R). DAC HYP blockade of CD25 inhibits effector T cell activation, regulatory T cell expansion and survival, and activation-induced T-cell apoptosis. Because CD25 blockade reduces IL-2 consumption by effector T cells, it increases IL-2 bioavailability allowing for greater interaction with the intermediate-affinity IL-2R, and therefore drives the expansion of CD56bright natural killer (NK) cells. Furthermore, there appears to be a direct correlation between CD56bright NK cell expansion and DAC HYP efficacy in reducing relapses and MRI evidence of disease activity in patients with RMS in phase II and phase III double-blind, placebo- and active comparator-controlled trials. Therapeutic efficacy was maintained during open-label extension studies. However, treatment was associated with an increased risk of rare adverse events, including cutaneous inflammation, autoimmune hepatitis, central nervous system Drug Reaction with Eosinophilia Systemic Symptoms (DRESS) syndrome, and autoimmune Glial Fibrillary Acidic Protein (GFAP) alpha immunoglobulin-associated encephalitis. As a result, DAC HYP was removed from clinical use in 2018. The lingering importance of DAC is that its use led to a deeper understanding of the underappreciated role of innate immunity in the potential treatment of autoimmune disease.

Engineering human stellate cells for beta cell replacement therapy promotes in vivo recruitment of regulatory T cells.

Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of pancreatic β cells. One of the promising therapeutic approaches in T1D is the transplantation of islets; however, it has serious limitations. To address these limitations, immunotherapeutic strategies have focused on restoring immunologic tolerance, preventing transplanted cell destruction by patients’ own immune system. Macrophage-derived chemokines such as chemokine-ligand-22 (CCL22) can be utilized for regulatory T cell (Treg) recruitment and graft tolerance. Stellate cells (SCs) have various immunomodulatory functions: recruitment of Tregs and induction of T-cell apoptosis. Here, we designed a unique immune-privileged microenvironment around implantable islets through overexpression of CCL22 proteins by SCs. We prepared pseudoislets with insulin-secreting mouse insulinoma-6 (MIN6) cells and human SCs as a model to mimic naive islet morphology. Our results demonstrated that transduced SCs can secrete CCL22 and recruit Tregs toward ​the implantation site in vivo. This study is promising to provide a fundamental understanding of SC-islet interaction and ligand synthesis and transport from SCs at the graft site for ensuring local immune tolerance. Our results also establish a new paradigm for creating tolerable grafts for other chronic diseases such as diabetes, anemia, and central nervous system (CNS) diseases, and advance the science of graft tolerance.

RILA blood biomarker as a predictor of radiation-induced sarcoma in a matched cohort study.

PurposeRadiation-induced sarcoma (RIS) is a rare but serious event. Its occurrence has been discussed during the implementation of new radiation techniques and justified appropriate radioprotection requirements. New approaches targeting intrinsic radio-sensitivity have been described, such as radiation-induced CD8 T-lymphocyte apoptosis (RILA) able to predict late radio-induced toxicities. We studied the role of RILA as a predisposing factor for RIS as a late adverse event following radiation therapy (RT).Patients and methodsIn this prospective biological study, a total of 120 patients diagnosed with RIS were matched with 240 control patients with cancer other than sarcoma, for age, sex, primary tumor location and delay after radiation. RILA was prospectively assessed from blood samples using flow cytometry.ResultsThree hundred and forty-seven patients were analyzed (118 RIS patients and 229 matched control patients). A majority (74%) were initially treated by RT for breast cancer. The mean RT dose was comparable with a similar mean (± standard deviation) for RIS (53.7 ± 16.0 Gy) and control patients (57.1 ± 15.1 Gy) (p = .053). Median RILA values were significantly lower in RIS than in control patients with respectively 18.5% [5.5–55.7] and 22.3% [3.8–52.2] (p = .0008). Thus, patients with a RILA >21.3% are less likely to develop RIS (p < .0001, OR: 0.358, 95%CI [0.221–0.599].ConclusionRILA is a promising indicator to predict an individual risk of developing RIS. Our results should be followed up and compared with molecular and genomic testing in order to better identify patients at risk. A dedicated strategy could be developed to define and inform high-risk patients who require a specific approach for primary tumor treatment and long term follow-up.

The MEK-ERK-MST1 Axis Potentiates the Activation of the Extrinsic Apoptotic Pathway during GDC-0941 Treatment in Jurkat T Cells.

The discrete activation of individual caspases is essential during T-cell development, activation, and apoptosis. Humans carrying nonfunctional caspase-8 and caspase-8 conditional knockout mice exhibit several defects in the progression of naive CD4+ T cells to the effector stage. MST1, a key kinase of the Hippo signaling pathway, is often presented as a substrate of caspases, and its cleavage by caspases potentiates its activity. Several studies have focused on the involvement of MST1 in caspase activation and also reported several defects in the immune system function caused by MST1 deficiency. Here, we show the rapid activation of the MEK-ERK-MST1 axis together with the cleavage and activation of caspase-3, -6, -7, -8, and -9 after PI3K signaling blockade by the selective inhibitor GDC-0941 in Jurkat T cells. We determined the phosphorylation pattern of MST1 using a phosphoproteomic approach and identified two amino acid residues phosphorylated in an ERK-dependent manner after GDC-0941 treatment together with a novel phosphorylation site at S21 residue, which was extensively phosphorylated in an ERK-independent manner during PI3K signaling blockade. Using caspase inhibitors and the inhibition of MST1 expression using siRNA, we identified an exclusive role of the MEK-ERK-MST1 axis in the activation of initiator caspase-8, which in turn activates executive caspase-3/-7 that finally potentiate MST1 proteolytic cleavage. This mechanism forms a positive feed-back loop that amplifies the activation of MST1 together with apoptotic response in Jurkat T cells during PI3K inhibition. Altogether, we propose a novel MEK-ERK-MST1-CASP8-CASP3/7 apoptotic pathway in Jurkat T cells and believe that the regulation of this pathway can open novel possibilities in systemic and cancer therapies.

S100A9-induced overexpression of PD-1/PD-L1 contributes to ineffective hematopoiesis in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are characterized by dysplastic and ineffective hematopoiesis that can result from aberrant expansion and activation of myeloid-derived suppressor cells (MDSCs) within the bone marrow (BM) niche. MDSCs produce S100A9, which mediates premature death of hematopoietic stem and progenitor cells (HSPCs). The PD-1/PD-L1 immune checkpoint impairs immune responses by inducing T-cell exhaustion and apoptosis, but its role in MDS is uncharacterized. Here we report an increased expression of PD-1 on HSPCs and PD-L1 on MDSCs in MDS versus healthy donors, and that this checkpoint is also activated in S100A9 transgenic (S100A9Tg) mice, and by treatment of BM mononuclear cells (BM-MNC) with S100A9. Further, MDS BM-MNC treated with recombinant PD-L1 underwent cell death, suggesting that the PD-1/PD-L1 interaction contributes to HSPC death in MDS. In accordance with this notion, PD-1/PD-L1 blockade restores effective hematopoiesis and improves colony-forming capacity in BM-MNC from MDS patients. Similar findings were observed in aged S100A9Tg mice. Finally, we demonstrate that c-Myc is required for S100A9-induced upregulation of PD-1/PD-L1, and that treatment of MDS HSPCs with anti-PD-1 antibody suppresses the expression of Myc target genes and increases the expression of hematopoietic pathway genes. We conclude anti-PD-1/anti-PD-L1 blocking strategies offer therapeutic promise in MDS in restoring effective hematopoiesis.

PD-1/PD-L1 blockade rescue exhausted CD8+ T cells in gastrointestinal stromal tumours via the PI3K/Akt/mTOR signalling pathway.

ObjectivesAlthough targeted therapy has revolutionized the treatment of gastrointestinal stromal tumours (GIST), it is almost never curative in GIST, and resistance commonly develops. One potential strategy is to combine targeted therapy with immunotherapy.Materials and methodsWe first studied Programmed cell death 1 ligand 1 (PD‐L1) expression and tumour‐infiltrating T cells (TILs) in GIST. IFN‐γ was used to induce the upregulation of PD‐L1 expression in GIST‐882 cells, a well‐known GIST cell line. CD8+ T‐cell apoptosis was determined by flow cytometry. The PI3K/Akt/mTOR levels in CD8+ T cells were examined by Western blotting.ResultsPD‐L1 expression was an independent factor of poor prognosis in GIST and resulted in exhausted T cells in the TILs population or the blood. Then, we found that PD‐L1 blockade alone could not increase tumour cell apoptosis in GIST. The apoptosis rate of CD8+ T cells was higher when T cells were cultured with PD‐L1+ GIST‐882 cells (GIST‐882 cells with high PD‐L1 expression) than when T cells were cultured with control GIST‐882 cells. However, when the PD‐L1 blockade was used, the apoptosis rates of the CD8+ T cells in the two groups became similar. Then, Western blotting showed the PI3K/Akt/mTOR levels of the CD8+ T cells rescued by the PD‐1/PD‐L1 blockade were higher than those of the CD8+ T cells not treated with the PD‐1/PD‐L1 blockade.ConclusionsPD‐L1 expression was an independent poor prognosis factor in GIST. PD‐1/PD‐L1 blockade rescued exhausted CD8+ T cells in GIST via the PI3K/Akt/mTOR signalling pathway. In GIST, PD‐1/PD‐L1 not only function as predictive biomarkers but also improve current therapies as treatment targets.

Abstract A210: Tumor-associated ADAM10 and ADAM17 produce soluble PD-L1 (sPD-L1, sB7-H1) and affect downstream tumor immunity – A resistance mechanism to PD-1 checkpoint blockade in melanoma

Abstract Background: Tumor surface PD-L1 (B7-H1) signals through T-cell surface receptor PD-1 to suppress antitumor immunity. PD-1 inhibitors like pembrolizumab block this pathway to prevent tumor escape from immunosurveillance. These PD-1 checkpoint inhibitors achieve objective response in 20-30% of patients with melanoma (1-3). As the majority of these patients are resistant to this treatment, there is a critical need to combat PD-1 checkpoint inhibitor resistance in melanoma. What is the mechanism of PD-1 checkpoint inhibitor resistance in melanoma? Our group has reported that soluble PD-L1 (sPD-L1, sB7-H1) is elevated in the serum of patients with PD-1 inhibitor-resistant melanoma and non-small cell lung cancer (NSCLC) (4). We have also shown that soluble PD-L1 is produced by cleavage from other tumor types and leads to apoptosis in CD8+ T-cells (5). The aim of this study is to confirm the source of sPD-L1 in human melanoma and its relation to outcomes. Methods: B7H1-expressing transgenic melanoma cells (Mel-B7H1) were treated with an array of protease inhibitors over 24 hours. Cells were then pelleted and supernatants were analyzed by ELISA for sPD-L1 (sB7-H1) production. Tumor serum samples were also analyzed by ELISA for sPD-L1 production.Tumor pathology slides from patients with melanoma (serum donors from the above study) were stained by immunohistochemistry with antibodies against proteases ADAM10 or ADAM17. Slides were visualized by microscopy for the presence of these antigens. Activated human CD8+ T-cells were cultured with recombinant human B7-H1 (PD-L1) protein, sPD-L1-rich, or sPD-L1-depleted supernatants from a tumor cell line in the presence of placebo or PD-1 checkpoint inhibitor antibodies. Cell survival was measured at 48 hours by flow cytometry.Results: ADAM10 inhibitor GI254023X, ADAM17 inhibitor TAPI-0, and broad metalloprotease inhibitor TAPI-2 inhibited Mel-B7H1 production of sB7H1 (p&amp;lt;0.001, 0.095, and 0.001 respectively). Specific inhibitors of other metalloproteases did not significantly alter sB7H1 production. PD-L1 transcription levels were not significantly altered. Tumor pathology slides from patients with high serum sPD-L1 exhibited high ADAM10 and/or ADAM17 staining. Conversely, tumors from patients with low-sPD-L1levels tended to exhibit low ADAM10 and/or ADAM17 staining. Recombinant PD-L1 and PD-L1-rich supernatants induced cell death of CD8+ T-cells, whereas PBS control solution and PD-L1-depleted supernatants did not. Supraphysiologic doses of pembrolizumab decreased cell death in PD-L1-rich supernatant-treated CD8+ T-cells. Conclusions: Matrix metalloprotease inhibitors ADAM10 and ADAM17 mediate sPD-L1 production from melanoma cells. In samples from patients with melanoma, immunohistochemical staining of these proteases in tumor tissue correlates with serum sPD-L1 levels. As we have shown previously, sPD-L1 induces apoptosis of activated CD8+ T-cells, which may explain PD-1 inhibitor resistance in most patients with melanoma.We posit that sPD-L1 outcompetes PD-1 inhibitors to suppress tumor immunosurveillance. Our results suggest that the combination of ADAM10/17 inhibitor and PD-1 checkpoint inhibitor may overcome this mechanism of PD-1 resistance in melanoma and other cancers. Future studies will determine (A) how a combined ADAM10/17 inhibitor and PD-1 inhibitor may function in clinical use, (B) how tumor-derived sPD-L1 signals downstream to cause apoptosis in T-cells, and (C) whether surface ADAM10/17 predict surface PD-L1 staining in melanoma.