T-cell Apoptosis Research Articles

Abstract 2517: Characterization of the immune microenvironment of Epstein-Barr virus associated gastric cancer

Abstract Consistent with better clinical outcome, Epstein-Barr Virus associated gastric cancer (EBVaGC) displays a particular expression pattern of immunological genes and is highly infiltrated by effector immune cells. To decipher potential mechanisms linking tumor infiltrating lymphocytes (TILs) with an expression of immune-related genes in EBVaGC we utilized RNAseq gene expression data from The Cancer Genome Atlas (TCGA) Stomach Adenocarcinoma (STAD) dataset and performed cellular deconvolution analyses by a mixed approach including two complementary approaches (MIXTURE and xCell). In accordance with previous studies, we observed an increase in CD8+ cells and Interferon activity. Further analysis by gene set variation analysis (GSVA) showed an increased exhausted CD8+ signature accompanied by a higher intratumoral Treg signature. Accordingly, an enrichment in both stimulatory and inhibitory immune checkpoint levels on EBVaGC tumors was observed. We next performed differential expression analysis comparing EBVaGC to other STAD subtypes (CIN, GS, HM-SNV, HM-indel) individually. As a result, from this approach, we generated a list of unique EBVaGC-specific genes that was used to construct EBVaGC-specific cell-cell interaction networks. To infer cell-cell interactions underlying the specific tumor immune microenvironment, we implemented the NicheNetR algorithm by including only differentially expressed ligands and targets from the list of EBVaGC genes obtaining a ligand-interaction matrix from which we constructed a EBVaGC-like gene signature. Functional annotation of this ligand-target network showed ligands and targets principally involved in chemotaxis, T-cell apoptosis, and cellular responses to bacterial infection. This signature correlated with better survival not only in EBVaGC but also in other TCGA cohorts such as colon adenocarcinoma and kidney renal clear cell carcinoma. Our findings identified an increase in exhausted CD8+ signature, high intratumoral Treg signature and enrichment in inhibitory immune checkpoint transcripts. We herein propose a new EBVaGC-like gene expression signature that correlates with better survival in multiple cancer types. In-vitro and in-vivo assays will be required for functional validation of these findings as well as clinical trials to confirm the role of our novel EBVaGC-like gene signature in a precision oncology setting. Grant Support: ANID/FONDECYT/POSTDOCTORADO/3201028, CONICYT-FONDAP 15130011, FONDECYT 1191928 & 1180241, IMII P09/016-F, CONICET and Universidad Católica de Córdoba 80020180100029CC, grant 33620180100993CB from the Universidad Nacional de Córdoba. Citation Format: Keila E. Torres, Charlotte N. Hill, Dario Rocha, Elmer Fernández, Ignacio A. Wichmann, Gareth I. Owen, Alejandro H. Corvalan. Characterization of the immune microenvironment of Epstein-Barr virus associated gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2517.

Abstract 2513: MDSC-derived TNF is a novel regulator of T-cell dysfunction in pancreatic cancer

Abstract Introduction: Abundance of myeloid-derived suppressor cells (MDSC) and a dysfunctional T-cell compartment are defining hallmarks of therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC). Using congenic in vivo murine models to phenocopy extremes of T-cell enrichment or exclusion, we sought to interrogate central MDSC-mediated mechanisms that govern immune tolerance in PDAC. Methods: Orthotopically implanted T-cell-excluded (Tcelllo) vs T-cell-enriched (T-cellhi) congenic KPC tumors, and intratumoral Ly6G+F4/80- MDSCs from both clones, were subjected to RNA sequencing. Ex vivo co-cultures evaluated the effects of intratumoral MDSC on splenic T-cells. Orthotopically injected KPC-T-celllo mice were treated with etanercept vs. vehicle, and immunophenotyping via flow cytometry was performed. Results: RNA-seq of KPC T-celllo vs. T-cellhi tumors revealed enrichment of myeloid immunoregulatory pathways, and downregulation of leukocyte activation/cytotoxicity pathways. Flow cytometry revealed a dramatic increase in MDSCs infiltrating KPC-Tcelllo tumors (P<0.001). To decipher MDSC-intrinsic mechanisms associated with T-cell exclusion, RNA-seq of MDSCs infiltrating T-cellhi clones revealed relative downregulation of MAPK signaling, and cytokine profiling of MDSCs conditioned with MAPK inhibitor trametinib revealed marked reduction in TNF secretion. Confocal microscopy confirmed striking decrease in TNF in MDSCs isolated from KPC- T-cellhi vs. Tcelllo tumors. Ex vivo MDSC-T-cell co-cultures significantly attenuated T-cell proliferation and activation (via IFN-γ release) while favoring T-cell apoptosis, which could be rescued by pre-conditioning MDSCs with either etanercept (TNFR2 decoy receptor) or MAPK pathway inhibitors. Orthotopically injected KPC-T-celllo tumor-bearing mice treated with etanercept demonstrated a remodeled TME vs. vehicle-treated mice, with attenuation in MDSC trafficking, enrichment in CD4+/CD8+ T-cell infiltration, and reduction in T-cell exhaustion. Conclusion: MDSC-derived TNF regulates T-cell dysfunction in PDAC via a MAPK-dependent mechanism. Compartment-specific inhibition of TNF may be a provocative strategy to overcome immune tolerance in PDAC. Citation Format: Anna Bianchi, Iago De Castro Silva, Nilesh U. Deshpande, Siddharth Mehra, Samara Singh, Austin R. Dosch, Vanessa T. Garrido, Christine I. Rafie, Nagaraj Nagathihalli, Nipun Merchant, Jashodeep Datta. MDSC-derived TNF is a novel regulator of T-cell dysfunction in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2513.

Granulocyte-Macrophage Colony-Stimulating Factor Modulates Myeloid-Derived Suppressor Cells and Treg Activity in Decompensated Cirrhotic Patients With Sepsis.

BackgroundDecompensated cirrhosis patients are more prone to bacterial infections. Myeloid-derived suppressor cells (MDSCs) expand in sepsis patients and disrupt immune cell functions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy helps in restoring immune cell functions and resolving infections. Its role in MDSC modulation in cirrhosis with sepsis is not well understood.MethodsA total of 164 decompensated cirrhotic—62 without (w/o), 72 with sepsis, and 30 with sepsis treated with GM-CSF—and 15 healthy were studied. High-dimensional flow cytometry was performed to analyze MDSCs, monocytes, neutrophils, CD4 T cells, and Tregs at admission and on days 3 and day 7. Ex vivo co-cultured MDSCs with T cells were assessed for proliferation and apoptosis of T cells and differentiation to Tregs. Plasma factors and mRNA levels were analyzed by cytokine-bead assay and qRT-PCR.ResultsFrequencies of MDSCs and Tregs were significantly increased (p = 0.011 and p = 0.02) with decreased CD4 T cells (p = 0.01) in sepsis than w/o sepsis and healthy controls (HCs) (p = 0.000, p = 0.07, and p = 0.01) at day 0 and day 7. In sepsis patients, MDSCs had increased IL-10, Arg1, and iNOS mRNA levels (p = 0.016, p = 0.043, and p = 0.045). Ex vivo co-cultured MDSCs with T cells drove T-cell apoptosis (p = 0.03, p = 0.03) with decreased T-cell proliferation and enhanced FOXP3+ expression (p = 0.044 and p = 0.043) in sepsis compared to w/o sepsis at day 0. Moreover, blocking the MDSCs with inhibitors suppressed FOXP3 expression. GM-CSF treatment in sepsis patients significantly decreased MDSCs and FOXP3+ Tregs but increased CD4 T-cell functionality and improved survival.ConclusionMDSCs have an immunosuppressive function by expanding FOXP3+ Tregs and inhibiting CD4+ T-cell proliferation in sepsis. GM-CSF treatment suppressed MDSCs, improved T-cell functionality, and reduced Tregs in circulation.

Decoy Receptor 3 Suppresses T-Cell Priming and Promotes Apoptosis of Effector T-Cells in Acute Cell-Mediated Rejection: The Role of Reverse Signaling

BackgroundDecoy receptor 3 (DcR3) belongs to the tumor necrosis factor (TNF) receptor superfamily and neutralizes TNF ligands, including FasL and TRAIL, to prevent T activation during T-cell priming. However, the cellular mechanisms underlying acute cell-mediated rejection (ACMR) remain unknown.MethodsWe generated DcR3 transgenic (Tg) mice and mice with high DcR3 expression (HDE) to study both in vivo and in vitro. FasR RNA knockdown in immortalized CD4+CD8+ T-cells was used to survey the role of DcR3 on FasR/Fas-associated protein with death domain (FADD)/caspase 8 pathway and its cross-link to TNF receptor-associated factor 1 (TNFR1)-associated death domain protein (TRADD) in suppressing TNFR1. TNF/TRADD knockout mice were used to show the importance of TNF adaptor protein.ResultsDcR3.Fc suppressed C57BL/6 female T-cell activation and transformation into CD4+CD69+, CD4+CD44+, and CD4+CD25+Foxp3+ when compared with isotype IgG1 and its co-treatment with FasL/TRAIL after exposing to bone marrow-derived dendritic cells (BMDCs) that carried alloantigen with male H-Y and minor antigenic determinant. Interleukin-17 and interferon-γ productions by BMDC-activated T-cells were lowered after co-treating with DcR3.Fc. DcR3.Fc induced effector T-cells (Teffs) and was susceptible to FasR-mediated apoptosis through the FADD/TRADD/caspase 8 pathway. After exposing to DcR3.Fc, TRADD was silenced, likely turning down the inflammatory response. The systemic effects of DcR3 Tg mice and HDE phenotype induced by the promoter of cytomegalovirus not only attenuated ACMR severity but also ameliorated the high serum creatinine and blood urea nitrogen levels even with high T-cell exposure frequencies. Besides this, DcR3 has minor biological effects on both MHC-matched and MHC-mismatched models.ConclusionsHigh DcR3 doses protect renal tubular epithelial cells from acute T-cell attack during the T-cell priming stage via interfering with TNF ligand-mediated reverse signaling and possibly promoting Teff apoptosis through FasR upregulation. Our findings supported that the decoy receptor is involved in T-cell modulation in kidney transplant rejection.

FAP-α+ immunofibroblasts in oral lichen planus promote CD4+ T-cell infiltration via CCL5 secretion.

Oral lichen planus (OLP) is a T cell-mediated, chronic inflammatory disease. CD4+ T-cell infiltration plays a crucial role in the pathogenesis of OLP. Fibroblasts are activated under pathological conditions and perform various functions. This study was designed to explore the immune activation and biological functions of OLP fibroblasts on CD4+ T cells. We detected the expression of fibroblast activation protein-alpha (FAP-α) in the oral tissues of patients with OLP and healthy controls using immunohistochemistry. Furthermore, expression of FAP-α and C-C motif chemokine ligand 5 (CCL5) in fibroblasts isolated from oral tissues of patients with OLP and healthy controls was assayed by quantitative PCR, Western blotting and enzyme-linked immunosorbent assay. Moreover, we assessed the effects of fibroblasts on CD4+ T-cell proliferation, apoptosis and migration using flow cytometry and Transwell assays. We found that FAP-α expression in the oral tissues of patients with OLP was significantly higher than that in healthy controls. FAP-α and CCL5 expression levels were significantly upregulated in OLP fibroblasts. Moreover, OLP fibroblasts promoted CD4+ T-cell proliferation and migration and inhibited CD4+ T cell apoptosis. In summary, our findings indicate that OLP fibroblasts are immunologically activated and induce CD4+ T-cell infiltration in OLP.

Study of the Role of Fas-Mediated Apoptosis of Peripheral Blood T-Lymphocytes in the Pathogenesis of Type 1 Diabetes Mellitus

Study of the Role of Fas-Mediated Apoptosis of Peripheral Blood T-Lymphocytes in the Pathogenesis of Type 1 Diabetes Mellitus

Novel, small molecule inhibitors of PD-1/PD-L1 pathway.

2597 Background: Programmed Cell Death 1 (PD-1) protein plays a key role in inhibiting immune responses and enhancing self-tolerance via modulation of T-cell activity, inducing T-cell apoptosis and inhibiting apoptosis of regulatory T cells. PD-L1 also plays an important role in various malignancies where it can attenuate the host immune response to tumor cells thereby favouring tumor progression and metastasis. High expression of PD-L1 in glioblastoma tumor tissues is associated with poor survival of patients, and PD-L1 may act as a prognostic predictor and an effective therapeutic target for glioblastoma. A number of monoclonal antibodies targeting PD-1/PD-L1 have approved for various malignancies. Still, efficacy of these antibodies in glioblastoma and brain metastasis continues to be moderate potentially owing to lack of or poor brain penetrance of these agents. Therefore, there is still a need for potent, selective small molecule PD-1/PD-L1 inhibitors with enhanced brain penetration in the treatment of such cancers. Methods: Rational design approaches were used to design novel small molecule PD-1/PD-L1 pathway inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Checkpoints signalling reporter assays as well cell based PD-L1 dimerization assays were used to assess the mechanistic and functional effects. In vivo efficacy was assessed in orthotopic GBM as well as in syngeneic and humanized subcutaneous tumor models in mice. Results: Our lead PD-L1 inhibitor JBI-2174 showed strong in vitro IC50 of ̃1 nM in TR-FRET assay that measures interaction between hPD-1 and hPD-L1 and a picomolar IC50 against monkey PD-L1. In selectivity assays for immunooncology targets, JBI-2174 was highly selective for PD-L1. JBI-2174 also inhibited PD-L1/PD-1 mediated signalling essential for T-cell modulation. JBI-2174 induced dimerization of PD-L1 as observed by size exclusion chromatography, which was confirmed by co-crystal structure and recapitulated in cell based dimerization assay. Elucidation of the co-crystal structure, clearly demonstrated that JBI-2174 clearly interacts with multiple amino acids on PD-L1 that are critical for PD-1 binding. JBI-2174 showed excellent oral bioavailability across pre-clinical species and sustained brain exposure. In the in vivo efficacy studies, JBI-2174 showed comparable efficacy to the anti-PD-L1 antibody or Atezolizumab in syngeneic (4T1, CT-26) and in partially humanized models (MC-38/hPD-L1). Further, oral administration of JBI-2174 resulted in statistically significant increase in survival (Day 27 in control vs day 38 in treated, p < 0.05) in a mouse glioma orthotopic model. Conclusions: The oral bioavailability and brain exposure of this molecule will make it attractive for cancers with unmet medical needs such as GBM and brain metastasis. IND enabling studies are being initiated for this compound.

The Love-Hate Relationship Between TGF-β Signaling and the Immune System During Development and Tumorigenesis

Since TGF-β was recognized as an essential secreted cytokine in embryogenesis and adult tissue homeostasis a decade ago, our knowledge of the role of TGF-β in mammalian development and disease, particularly cancer, has constantly been updated. Mounting evidence has confirmed that TGF-β is the principal regulator of the immune system, as deprivation of TGF-β signaling completely abrogates adaptive immunity. However, enhancing TGF-β signaling constrains the immune response through multiple mechanisms, including boosting Treg cell differentiation and inducing CD8+ T-cell apoptosis in the disease context. The love-hate relationship between TGF-β signaling and the immune system makes it challenging to develop effective monotherapies targeting TGF-β, especially for cancer treatment. Nonetheless, recent work on combination therapies of TGF-β inhibition and immunotherapy have provide insights into the development of TGF-β-targeted therapies, with favorable outcomes in patients with advanced cancer. Hence, we summarize the entanglement between TGF-β and the immune system in the developmental and tumor contexts and recent progress on hijacking crucial TGF-β signaling pathways as an emerging area of cancer therapy.

Characteristics of cellular and humoral immunity parameters in patients with mechanical jaundice, caused by cholangiocarcinoma

In the modern world, obstructive jaundice caused by developing a malignant disease cholangiocarcinoma, poses a significant medical and social problem in economically developed and in developing countries. The aim of this study was to examine parameters of cellular and humoral immunity in patients with obstructive jaundice caused by cholangiocarcinoma before and after surgical intervention. There were enrolled 56 patients with obstructive jaundice caused by cholangiocarcinoma (stages T2-3N0-1M0) and 90 age-matched apparently healthy volunteers. Cellular immunity parameters were studied by using an FC500 flow cytometer (Beckman Coulter, USA). The parameters of humoral immunity were assessed by measuring level of serum immunoglobulins A, M, G, E by using enzyme-linked immunosorbent assay kits manufactured by Vector-Best (Russia). The results were statistically analyzed using the Statistica v. 12.0 software (StatSoft Inc., USA). According to the study results, in patients with obstructive jaundice caused by cholangiocarcinoma, there was revealed activation of humoral immunity due to increased serum level of IgA, IgG, IgE with a profoundly depressed some arms of cellular immunity contributing to the imbalanced work of the entire immune system. The imbalance in the cellular arm of immune system was manifested by decreased count of T helper and increased cytotoxic T cell subsets as well as decline in the CD4+/CD8+ T cell ratio. An increased count of cytotoxic T lymphocytes points at lowered T cell immunity and existence of a cytotoxic effect at the cellular level. A decrease in the count of pan-marker-positive T lymphocytes indicates about generally reduced activity of the T cell arm due to altered T cell function and, as a result, affected antigen-presenting function in immune cells. The development of T cell immunodeficiency due to T-cell apoptosis confirms upregulated expression of immunomarkers CD38+, CD95+. An increased serum level for some immunoglobulins suggests developing imbalance in the humoral immunity, whereas rise in the circulating immune complexes indicates about pronounced body intoxication caused by obstructive jaundice due to hyperbilirubinemia and disintegration of tumor cells. Application of surgical correction of obstructive jaundice contributed to normalizing parameters of cellular and humoral immunity, resulting in immunocorrective effect.

Electroacupuncture at ST36 (Zusanli) Prevents T-Cell Lymphopenia and Improves Survival in Septic Mice.

PurposeSepsis is the main cause of death in intensive care unit. Maladaptive cytokine storm and T-cell lymphopenia are critical prognosis predictors of sepsis. Electroacupuncture (EA) is expected to be an effective intervention to prevent sepsis. This study aims to determine the potential of EA at ST36 (Zusanli) to prevent experimental septic mice.MethodsMice were randomly assigned into PBS, LPS, or EA+LPS group. EA (0.1 mA, continuous wave, 10 Hz) was performed stimulating the ST36 for 30 min, once a day for 3 days. After the third day, all mice were challenged with PBS or LPS (4 mg/kg) simultaneously. Mice were evaluated for survival, ear temperature, and other clinical symptoms. Lung and small intestine tissue injuries were analyzed by hematoxylin and eosin staining. Bio-Plex cytokine assay was used to analyze the concentration of cytokines. T lymphocytes were analyzed by flow cytometry and Western blot assays. The role of T cells in preventing sepsis by EA was analyzed by using nude mice lacking T lymphocytes.ResultsEA at ST36 improved survival, symptom scores, and ear temperature of endotoxemic mice. EA also improved dramatically pulmonary and intestinal injury by over 50% as compared to untreated mice. EA blunted the inflammatory cytokine storm by inducing a lasting inhibition of the production of major inflammatory factors (TNF-α, IL-1β, IL-5, IL-6, IL-10, IL-17A, eotaxin, IFN-γ, MIP-1β and KC). Flow cytometry and Western blot analyses showed EA significantly reduced T-lymphocyte apoptosis and pyroptosis. Furthermore, T lymphocytes were critical for the effects of EA at ST36 stimulation blunted serum TNF-α levels in wild-type but not in nude mice.ConclusionEA halted systemic inflammation and improved survival in endotoxemic mice. These effects are associated with the protective effect of EA on T lymphocytes, and T cells are required in the anti-inflammatory effects of EA in sepsis.

MRNA Expression of Genes Involves In Intrinsic Apoptotic Pathway During HIV Infection and Therapy and Its Relation In Circulating cd4 T-Lymphocytes

Background: CD4 T-lymphocyte apoptosis in Human Immunodeficiency Virus infection may occur due to activation of the intrinsic apoptotic pathway or triggered via death receptors. In this study, the differential expression of the genes involved in the intrinsic apoptotic pathway was investigated on circulating CD4 T-lymphocytes of HIV-infected patients and healthy controls. Methods: The study included 55 HIV patients who were (1) asymptomatic antiretroviral therapy naive, (2) symptomatic antiretroviral therapy naive, and (3) on antiretroviral therapy. As control 20 healthy persons were included. CD4 T-lymphocytes were isolated from blood samples of HIV patients and healthy controls using density gradient centrifugation method and by negative selection from peripheral blood mononuclear cells (using CD4+ T Cell Isolation Kit followed by total RNA extraction and cDNA synthesis. Genes involved in the intrinsic apoptotic pathway of CD4+ T-lymphocyte including BAX, BAK, BIM, MCL-1 and BCL-2, Caspase-3, Caspase-9 and Calpain-1 were selected and analyzed by real-time PCR to detect their expression. The fold changes of gene expression of PLHIV groups were compared with that of healthy controls. Results: Among the genes under study, no significant upregulation in the expression of BAX, BAK, Caspase-3, Calpain-1 and no significant downregulation in BIM, MCL-1 expression was observed with the exception of upregulation of BCL-2 and downregulation of Caspase-9 expression which were statistically significant (p <0.05). None of the studied genes showed any significant correlations between the gene expressions and CD4 T-lymphocyte count. Conclusion: Gene expression of intrinsic apoptotic pathway may not have significant effect on peripheral CD4 T-lymphocytes count in HIV infection and treatment. Bangladesh J Medicine 2022; 33: 176-185

MTOR deletion ameliorates CD4 + T cell apoptosis during sepsis by improving autophagosome-lysosome fusion

Autophagy dysfunction contributes to CD4 + T cell apoptosis during sepsis leading to impairment of adaptive immunity. However, the underlying mechanism is unclear. The mammalian target of rapamycin (mTOR) pathway modulates CD4 + T cell survival during sepsis through mechanisms that are not fully understood. We developed a mouse model of sepsis through cecal ligation and puncture (CLP) to investigate dynamic changes in autophagy in CD4 + T cells. We used T cell specific-mTOR/tuberous sclerosis complex 1 (TSC1)-knockout mice to explore the roles of the mTOR pathway in modulating autophagy during sepsis. We observed reduced fusion of autophagosomes with lysosomes in the CD4 + T cells of CLP mice, which may represent a characteristic feature of autophagy dysfunction. Deletion of mTOR relieved autophagosome-lysosome fusion dysfunction and ameliorated apoptosis of CD4 + T cells in CLP mice, but this rescued phenotype was abolished by treatment with bafilomycin A1, a specific A-L fusion inhibitor. We further explored the underlying molecular mechanism and found that phosphorylation levels of transcription factor EB were significant higher in CLP mice and that expression of A-L fusion protein SNAREs were restricted, both of which were ameliorated by mTOR deletion. Taken together, these results suggest that the mTOR pathway plays a critical role in regulation of CD4 + T-cell apoptosis during sepsis, partly through regulation of A-L fusion-related protein transcription.

Assessment of the functions of cultured lymphocytes when exposed to drugs used in cosmetology

The aim of the study was to determine the number of lymphocytes, intracellular cytokines produced by lymphocytes, and the cell cycle of lymphocytes isolated from the blood of patients when exposed to various drugs, as well as to assess the functions of cultured lymphocytes when exposed to drugs in vivo and in vitro.Materials and methods. The study involved lymphocytes isolated from the blood of healthy women under various conditions. At the first stage of the study, T-lymphocytes were isolated from the blood of patients before exposure to the drug. The absolute and relative lymphocyte count, the number of intracellular cytokines, and the cell cycle were determined.At the second stage, the drugs were added to the nutrient medium, where lymphocytes isolated from the blood of patients who did not receive systemic drugs were cultured. The placental extract preparation was added to the lymphocytes isolated from the first group of patients, while the hyaluronic acid preparation was added to the lymphocytes isolated from the second group of patients.At the third stage, the lymphocytes isolated from the blood of patients after systemic exposure to the placental extract preparation or hyaluronic acid preparation were isolated and cultured, after which the same lymphocyte parameters were determined.Results. The number of T-lymphocytes increased with the systemic use of the placental extract and hyaluronic acid preparations and practically did not change compared with the baseline data, when these drugs were added to the nutrient medium. Placental extract and hyaluronic acid had a positive effect on the mitotic activity of cells; it is worth noting that the effect of placental extract was greater than that of hyaluronic acid. Both drugs did not have a negative effect on apoptosis of T-lymphocytes. Under the effect of placental extract, lymphocytes secreted more interleukins, which contributed to proliferation of keratinocytes.Conclusion. The placental extract and hyaluronic acid preparations have a stimulating effect on keratinocytes. The placental extract preparation has a stimulating effect on T-lymphocytes after systemic exposure of the body to it.

Mesenchymal stem cells empower T cells in the lymph nodes via MCP-1/PD-L1 axis

Mesenchymal stem cells (MSCs) are a type of immunosuppressive stromal cell found in multiple tissues and organs. However, whether MSCs possess immunosupportive characteristics remains unclear. In this study, we showed that the lymph nodes contain immunosupportive MSCs. They produce and secrete a high level of MCP-1 to promote T-cell proliferation and differentiation, in contrast to bone marrow MSCs (BMMSCs), which repress T-cell activation. Unlike BMMSCs, lymph node MSCs (LNMSCs) fail to respond to activated T-cell-induced production of PD-L1 to induce T-cell apoptosis. Mechanistically, MCP-1 activates phospho-Erk to sustain T-cell proliferation and activation while it represses NF-κB/PD-L1 pathway to avoid induction of T-cell apoptosis. Interestingly, inflammatory lymph node-derived LNMSCs abolish their immunosupportive function due to reduction of MCP-1 expression. Finally, we show that systemic infusion of LNMSCs rescues immunosuppression in cytoxan (CTX)-treated mice. This study reveals a previously unrecognized mechanism underlying MSC-based immunoregulation using the MCP-1/PD-L1 axis to energize T cells and suggests a potential to use MSCs to treat immunosuppressive disorders.

Population-based targeted RNA sequencing reveals novel disease-related gene fusions in pediatric and adult T-ALL

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a genetically diverse disease characterized by a heterogeneous profile of genetic lesions. Recent transcriptome studies identified a number of new T-ALL-related gene fusions. Novel gene fusions could be employed as disease-specific molecular markers and provide a better understanding of T-ALL biology, proving the need for further omics sequencing studies. MethodsWe performed a population-based analysis of 65 (26 pediatric and 39 adults) Lithuanian T-ALL patients. Targeted RNA sequencing (RNA-seq) was used to detect recurrent and novel T-ALL-related fusion transcripts and gene sequence variants. RT-qPCR was used to calculate the relative gene expression of fusion transcripts. ResultsWe identified disease-related gene alterations in 57/65 (87.7%) T-ALL cases, of which four patients harbored gene fusions that affect ABL-class or JAK-STAT signaling pathways. Five novel gene fusions were detected in 4/65 (6.2%) T-ALL cases: CD99::ABL2 and ZEB1::GNAS in the same case, CTCF::ENKD1, DIAPH1::JAK2, and CDK6::NEK1. Novel fusion transcripts encode chimeric proteins that can potentially affect T-cell proliferation, apoptosis, and help to maintain leukemic cells. Importantly, novel fusion transcripts were not detected in the clinical remission samples attributing these fusions to the leukemic compartment. ConclusionsWe report a similar incidence rate of recurrent gene alterations affecting T-ALL-related molecular signaling pathways in both Lithuanian T-ALL patients and other T-ALL studies. Our data suggest that newly identified gene fusions are specific to leukemic T-cells and provide new molecular insights on T-ALL pathogenesis. Further research is needed to confirm these findings.

Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis

ObjectiveInclusion body myositis (IBM) has an unclear molecular etiology exhibiting both characteristic inflammatory T-cell activity and rimmed-vacuolar degeneration of muscle fibers. Using in-depth gene expression and splicing studies, we aimed at understanding the different components of the molecular pathomechanisms in IBM.MethodsWe performed RNA-seq on RNA extracted from skeletal muscle biopsies of clinically and histopathologically defined IBM (n = 24), tibial muscular dystrophy (n = 6), and histopathologically normal group (n = 9). In a comprehensive transcriptomics analysis, we analyzed the differential gene expression, differential splicing and exon usage, downstream pathway analysis, and the interplay between coding and non-coding RNAs (micro RNAs and long non-coding RNAs).ResultsWe observe dysregulation of genes involved in calcium homeostasis, particularly affecting the T-cell activity and regulation, causing disturbed Ca2+-induced apoptotic pathways of T cells in IBM muscles. Additionally, LCK/p56, which is an essential gene in regulating the fate of T-cell apoptosis, shows increased expression and altered splicing usage in IBM muscles.InterpretationOur analysis provides a novel understanding of the molecular mechanisms in IBM by showing a detailed dysregulation of genes involved in calcium homeostasis and its effect on T-cell functioning in IBM muscles. Loss of T-cell regulation is hypothesized to be involved in the consistent observation of no response to immune therapies in IBM patients. Our results show that loss of apoptotic control of cytotoxic T cells could indeed be one component of their abnormal cytolytic activity in IBM muscles.

Cancer cell-intrinsic resistance to BiTE therapy is mediated by loss of CD58 costimulation and modulation of the extrinsic apoptotic pathway

BackgroundBispecific T-cell engager (BiTE) molecules induce redirected lysis of cancer cells by T cells and are an emerging modality for solid tumor immunotherapy. While signs of clinical activity have been...

Fibroblasts From Idiopathic Pulmonary Fibrosis Induce Apoptosis and Reduce the Migration Capacity of T Lymphocytes.

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease of unknown etiology. Myofibroblasts are organized in peculiar subepithelial fibroblasts foci (FF), where they abnormally persist and exclude lymphocytes by unclear mechanisms. FF are the source of an excessive extracellular matrix, which results in progressive stiffening and destruction of the lung architecture. We hypothesized that the absence of T cells inside the FF could be related, at least partially, to an inefficient function of lymphocytes induced by IPF fibroblasts. Here, we evaluated the effect of a supernatant from IPF fibroblasts on T-cell apoptosis and migration capacity. Data showed that IPF fibroblasts secrete pro-apoptotic molecules (both from extrinsic and intrinsic pathways), generating a microenvironment that induces apoptosis of T cells at 3 h of culture, despite a weak anti-apoptotic profile exhibited by these T cells. At 24 h of culture, the supernatants from both IPF and control fibroblasts provoked T-cell death. However, at this time of culture, IPF fibroblasts caused a marked decrease in T-cell migration; in contrast, control lung fibroblasts induced an increase of T-cell migration. The reduction of T-cell migratory capacity provoked by IPF fibroblasts was associated with a negative regulation of RHOA and ROCK, two essential GTPases for migration, and was independent of the expression of chemokine receptors. In conclusion, our findings demonstrate that IPF fibroblasts/myofibroblasts induce apoptosis and affect T-cell migration, revealing a mechanism involved in the virtual absence of T lymphocytes inside the FF.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-β receptor I versus II.

SUMMARYIntrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-β. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody. We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-β receptors I (TβRI) and II (TβRII). While the extracellular domain of PD-L1 (amino acids 19–238) targets TβRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TβRI mRNA from degradation by the RNA exosome complex. PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.

Association of Radiation-induced CD4 and CD8 T-Lymphocyte Apoptosis with Radiation-induced Fibrosis after Breast Conserving Therapy

Background: Around 70% of early breast cancer patients choose breast conserving therapy (BCT), with equal overall and disease-free survival rates compared to mastectomy. As a result, treatment decisions are currently guided by cosmetic outcomes and quality of life (QoL). Unfortunately, 13.4% of all BCT patients are affected by a grade 2 of radiation-induced fibrosis (RIF) of the breast. The individual risk of RIF could be predicted based the reduced radiation-induced lymphocyte apoptosis (RILA).