Chronic lymphocytic leukemia is marked by profound defects in T-cell function. Programmed death-1 is a receptor involved in tumor-mediated immunosuppression through binding of the PD-L1 ligand. ...Multiparametric flow cytometry and immunohistochemistry were used to study PD-1/PD-L1 expression. Functional assays were used to determine the involvement of the PD-1/PD-L1 axis in T-cell responses. PD-1 expression by CD4(+) and CD8(+) T lymphocytes was significantly higher in 117 chronic lymphocytic leukemia patients than in 33 donors of a comparable age. CD4(+) and CD8(+) T lymphocytes from chronic lymphocytic leukemia patients displayed increased numbers of effector memory and terminally differentiated cells, respectively, when compared to controls. The number of effector memory CD4(+) and terminally differentiated CD8(+) lymphocytes positively associated with a more advanced stage of disease, treatment requirements and unfavorable genomic aberrations. Furthermore, leukemic lymphocytes expressed higher levels of PD-L1 than circulating B lymphocytes from normal donors. PD-1 and PD-L1 surface expression spiked in proliferating T and B lymphocytes, suggesting that this interaction works efficiently in activated environments. Within chronic lymphocytic leukemia proliferation centers in the lymph node, CD4(+)/PD-1(+) T lymphocytes were found to be in close contact with PD-L1(+) chronic lymphocytic leukemia cells. Lastly, functional experiments using recombinant soluble PD-L1 and blocking antibodies indicated that this axis contributes to the inhibition of IFN-γ production by CD8(+) T cells. These observations suggest that pharmacological manipulation of the PD-1/PD-L1 axis may contribute to restoring T-cell functions in the chronic lymphocytic leukemia microenvironment.
Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike ...properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin IL 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL.
•CLL lymphocytes show high intracellular and extracellular NAMPT levels, further increased upon activation.•eNAMPT prompts differentiation of CLL monocytes into M2 macrophages that sustain CLL survival and reduce T-cell proliferation.
Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, ...the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.
The potential of dendritic cell (DC)-based immunotherapy to treat cancer is, nowadays, well documented. Still, the clinical success of immune checkpoint inhibitors has dampened the interest in ...anticancer DC vaccination. For highly life-threatening tumors that are regarded as nonimmunogenic, such as mesothelioma, however, T helper 1 immunity-biased DC-based immunotherapy could still represent an attractive strategy. In this study, we took advantage of photodynamic therapy (PDT) to induce immunogenic cell death to generate mesothelioma cell lysates for DC priming and evaluated such a vaccine to treat peritoneal mesothelioma. We found that the white light in vitro activation of the photosensitizer OR141 led to mesothelioma cell death, together with the release of bona fide danger signals that promote DC maturation. The administration of a PDT-based DC vaccine to mice bearing peritoneal mesothelioma led to highly significant survival when compared with sham or control animals treated with anti-CTLA4 antibodies. This was further supported by a strong CD8
and CD4
T cell response, characterized by an increased proliferation, cytotoxic activities and the expression of activation markers, including interferon gamma (IFN). Moreover, the PDT-based DC vaccine led to a significant increase in IFN
T cells infiltered within mesothelioma, as determined by flow cytometry and immunohistochemistry. Finally, in vivo tracking of intraperitoneally administered DCs led us to document rapid chemotaxis towards tumor-occupied lymphatics (vs. lipopolysaccharide (LPS)-treated DC). DCs pulsed with PDT-killed mesothelioma cells also exhibited a significant increase in CCR7 receptors, together with an intrinsic capacity to migrate towards the lymph nodes. Altogether, these results indicate that PDT-based DC vaccination is particularly suited to induce a potent immune response against peritoneal mesothelioma.
For poorly immunogenic tumors such as mesothelioma there is an imperious need to understand why antigen-presenting cells such as dendritic cells (DCs) are not prone to supporting the anticancer T ...cell response. The tumor microenvironment (TME) is thought to be a major contributor to this DC dysfunction. We have reported that the acidic TME component promotes lipid droplet (LD) formation together with epithelial-to-mesenchymal transition in cancer cells through autocrine transforming growth factor-β2 (TGF-β2) signaling. Since TGF-β is also a master regulator of immune tolerance, we have here examined whether acidosis can impede immunostimulatory DC activity. We have found that exposure of mesothelioma cells to acidosis promotes TGF-β2 secretion, which in turn leads to LD accumulation and profound metabolic rewiring in DCs. We have further documented how DCs exposed to the mesothelioma acidic milieu make the anticancer vaccine less efficient in vivo, with a reduced extent of both DC migratory potential and T cell activation. Interestingly, inhibition of TGF-β2 signaling and diacylglycerol O-acyltransferase (DGAT), the last enzyme involved in triglyceride synthesis, led to a significant restoration of DC activity and anticancer immune response. In conclusion, our study has identified that acidic mesothelioma milieu drives DC dysfunction and altered T cell response through pharmacologically reversible TGF-β2-dependent mechanisms.
The platelet transmembrane receptor GPVI can be assessed together with other platelet membrane markers in a whole blood multicolor flow cytometry panel. The advantage of combining multiple antibodies ...in a single tube is the possibility of distinguishing multiple platelet subgroups. In this short communication, we describe an activation problem encountered with anti-GPVI, clone HY101. Activation of platelets was seen after the addition of anti-GPVI in a flow cytometry panel, highlighted by the expression of the activation markers CD62P, PAC-1, CD63, and CD107a. This was also confirmed by platelet aggregation studies.
The study aimed to evaluate the impact of abdominal drain placement (vs. omission) on perioperative outcomes of robot-assisted partial nephrectomy (RAPN), focusing on complications, time to ...canalization, deambulation, and pain management. A prospectively-maintained institutional database was queried to get data of patients who underwent RAPN for renal masses between January 2018 and May 2023 at our Institution. Baseline, surgical, and postoperative data were collected. Retrieved patients were stratified based upon placement of abdominal drain (Y/N). Descriptive analyses comparing the two groups were conducted as appropriate.77 After adjusting for potential confounders, a logistic regression analysis was conducted to evaluate significant predictors of any grade and "major" complications. 342 patients were included: 192 patients in the "drain group" versus 150 patients in the "no-drain" group. Renal masses were larger (p < 0.001) and at higher complexity (RENAL score, p = 0.01), in the drain group. Procedures in the drain group had statistically significantly longer operative time, ischemia time, and higher blood loss (all p-values < 0.001). The urinary collecting system was more likely involved compared to the no-drain group (p = 0.01). At multivariate analysis, abdominal drainage was not a significant predictor of any grade (OR 0.79, 95%CI 0.33-1.87) and major postoperative complications (OR 3.62, 95%CI 0.53-9.68). Patients in the drain group experienced a statistically significantly higher hemoglobin drop (p < 0.01). Moreover, they exhibited statistically significant higher paracetamol consumption (p < 0.001) and need for additional opioids (p = 0.02). In summary, the study results suggest the safety of omitting drain placement and remark on the need for personalized decision-making, which considers patient and procedural factors.
Tobacco smoking is known to impact circulating levels of major immune cells populations, but its effect on specific immune cell subsets remains poorly understood. Here, using high-resolution data ...from 223 healthy women (25 current and 198 never smokers), we investigated the association between smoking status and 35,651 immune traits capturing immune cell subset frequencies. Our results confirmed that active tobacco smoking is associated with increased frequencies of circulating CD8+ T cells expressing the CD25 activation marker. Moreover, we identified novel associations between smoking status and relative abundances of CD8+ CD25+ memory T cells, CD8+ memory T cells expressing the CCR4 chemokine receptor, and CD4+CD8+ (double-positive) CD25+ T cells. We also observed, in current smokers, a decrease in the relative frequencies of CD4+ T cells expressing the CD38 activation marker and an increase in class-switched memory B cell isotypes IgA, IgG, and IgE. Finally, using data from 135 former female smokers, we showed that the relative frequencies of immune traits associated with active smoking are usually completely restored after smoking cessation, with the exception of subsets of CD8+ and CD8+ memory T cells, which persist partially altered. Our results are consistent with previous findings and provide further evidence on how tobacco smoking shapes leukocyte cell subsets proportion toward chronic inflammation.
Trastuzumab is a monoclonal antibody selectively directed against Her2 and approved for the treatment of Her2 overexpressing breast cancer patients. Its proposed mechanisms of action include ...mediation of antibody-dependent cellular cytotoxicity (ADCC) by triggering FcgammaRIII on natural killer (NK) cells. This study addresses the correlation between overall NK function and trastuzumab's clinical activity.
Clinical and immunological responses were assessed in 26 patients receiving trastuzumab monotherapy as maintenance management after chemotherapy (8 mg/kg load and then standard doses of 6 mg/kg every 3 weeks). Cytotoxic activity against the MHC class I-negative standard NK target K562 cell line and HER2-specific ADCC against a trastuzumab-coated Her2-positive SKBR3 cell line were assessed in peripheral blood mononuclear cells (PBMC) harvested after the first standard dose. After six months, seventeen patients were scored as responders and nine as non-responders according to the RECIST criteria, while Progression-Free Survival (PFS) was calculated during a 12 months follow-up.
The responders had significantly higher levels of both NK and ADCC activities (p < 0.05) that were not different from those of eleven normal controls. The NK activity of the non-responders was significantly (p < 0.05) lower than that of the normal controls. At twelve months, there was a marked correlation between PFS and NK activity only. PFS was significantly longer in patients with high levels of NK activity, whereas its pattern was unrelated to high or low ADCC activity.
One of the mechanisms of action of trastuzumab is NK cell-mediated ADCC lysis of the Her2-positve target cell. We show here that its potency is correlated with the short-term response to treatment, whereas longer protection against tumor expansion seems to be mediated by pure NK activity.
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