In high-grade ovarian carcinoma, platinum-sensitive tumor recurrence and acquired-resistance derive from quiescent residual cancer cells that overexpress CRYAB, CEACAM6, and SOX2.

Most high-grade ovarian carcinomas (HGOCs) are sensitive to carboplatin (CBP)-based chemotherapy but frequently recur within 24 months. Recurrent tumors remain CBP-sensitive and acquire resistance only after several treatment rounds. Recurrences arise from a small number of residual tumor cells not amenable to investigation in patients. We developed patient-derived xenografts (PDXs) that allow the study of these different stages of CBP-sensitive recurrence and acquisition of resistance. We generated PDX models from CBP-sensitive and intrinsically resistant HGOC. PDXs were CBP- or mock-treated and tumors were sampled, after treatment and at recurrence. We also isolated models with acquired-resistance from CBP-sensitive PDXs. Tumors were characterized at the histological and transcriptome levels. PDX models reproduced treatment response seen in the patients. CBP-sensitive residual tumors contained nonproliferating tumor cell clusters embedded in a fibrotic mesh. In nontreated PDX tumors and treated CBP-resistant tumors, fibrotic tissue was not prevalent. Residual tumors had marked differences in gene expression when compared to naïve and recurrent tumors, indicating downregulation of the cell cycle and proliferation and upregulation of interferon response and the epithelial-mesenchymal transition. This gene expression pattern resembled that described in embryonal diapause and 'drug-tolerant persister' states. Residual and acquired-resistance tumors share the overexpression of three genes: CEACAM6, CRYAB, and SOX2. Immunostaining analysis showed strong CEACAM6, CRYAB, and SOX2 protein expression in CBP-sensitive residual and acquired-resistance PDX, thus confirming the RNA profiling results. In HGOC PDX, CBP-sensitive recurrences arise from a small population of quiescent, drug-tolerant, residual cells embedded in a fibrotic mesh. These cells overexpress CEACAM6, CRYAB, and SOX2, whose overexpression is also associated with acquired resistance and poor patient prognosis. CEACAM6, CRYAB, and SOX2 may thus serve as a biomarker to predict recurrence and emergence of resistant disease in CBP-treated HGOC patients. © 2022 The Pathological Society of Great Britain and Ireland.

ADAM8 signaling drives neutrophil migration and ARDS severity.

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8-/- mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

STAT3 is over-activated within CD163pos bone marrow macrophages in both Multiple Myeloma and the benign pre-condition MGUS.

Tumour-associated macrophages (TAMs) support cancer cell survival and suppress anti-tumour immunity. Tumour infiltration by CD163pos TAMs is associated with poor outcome in several human malignancies, including multiple myeloma (MM). Signal transducer and activator of transcription 3 (STAT3) is over-activated in human cancers, and specifically within TAMs activation of STAT3 may induce an immunosuppressive (M2-like) phenotype. Therefore, STAT3-inhibition in TAMs may be a future therapeutic strategy.We investigated TAM markers CD163, CD206, and activated STAT3 (pSTAT3) in patients with MGUS (n = 32) and MM (n = 45), as well as healthy controls (HCs, n = 13).Blood levels of the macrophage biomarkers sCD163 and sCD206, and circulating cytokines, as well as bone marrow mRNA expression of CD163 and CD206, were generally increased in MGUS and MM patients, compared to HCs, but to highly similar levels. By immunohistochemistry, bone marrow levels of pSTAT3 were increased specifically within CD163pos cells in both MGUS and MM patients.In conclusion, macrophage-related inflammatory changes, including activation of STAT3, were present already at the MGUS stage, at similar levels as in MM. Specific increase in pSTAT3 levels within CD163pos cells supports that the CD163 scavenger receptor may be a useful target for future delivery of STAT3-inhibitory drugs to TAMs in MM patients.

Human and mouse muscle transcriptomic analyses identify insulin receptor mRNA downregulation in hyperinsulinemia-associated insulin resistance.

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have demonstrated that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome-wide level. Transcriptomic meta-analysis in >450 human samples demonstrated that fasting insulin reliably and negatively correlated with INSR mRNA in skeletal muscle. To establish causality and study the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated C2C12 myotubes with elevated insulin for 16 h, followed by 6 h of serum starvation, and established that acute AKT and ERK signaling were attenuated in this model of in vitro hyperinsulinemia. Global RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signaling, FOXO signaling, and glucose metabolism pathways indicative of 'hyperinsulinemia' and 'starvation' programs. Consistently, we observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Bioinformatic modeling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA (and JUND, MAX, and MXI as positive regulators of Irs2 mRNA). Together, our analysis identifies mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology and disease progression of type 2 diabetes.

Developing a pro-angiogenic placenta derived amniochorionic scaffold with two exposed basement membranes as substrates for cultivating endothelial cells.

Decellularized and de-epithelialized placenta membranes have widely been used as scaffolds and grafts in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique substrate which provides an enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which resulted in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in vivo pro-angiogenic capabilities of the substrate were also evaluated. Histological staining, immunohistochemistry (IHC) staining for collagen IV, and scanning electron microscope demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay, real-time polymerase chain reaction for endothelial adhesion molecules, and IHC demonstrated that both side basement membranes could support the growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided substrate with two exposed surfaces for cultivating various cells would have potential applications in the skin, cardiac, vascularized composite allografts, and microvascular tissue engineering.

The relationship between health-related quality of life and melancholic depressive symptoms is modified by brain insulin receptor gene network.

To investigate whether expression-based polygenic risk scores for the insulin receptor gene network (ePRS-IRs) modifiy the association between type of depressive symptoms and health-related quality of life (HRQoL). This cross-sectional study includes 1558 individuals from the Helsinki Birth Cohort Study. Between 2001 and 2004, the Short Form-36 questionnaire was employed to assess mental and physical components of HRQoL and Beck Depression Inventory (BDI) to assess depressive symptoms. Depressive symptoms were categorized into minimal (BDI < 10), non-melancholic and melancholic types of depression. The ePRS-IRs were calculated for the hippocampal (hePRS-IR) and the mesocorticolimbic (mePRS-IR) regions of the brain. General linear regression models adjusted for age, sex, population stratification, lifestyle factors and body mass index were applied to analyze the data. Both types of depressive symptoms were associated with lower HRQoL (p < 0.0001). HePRS-IR modified the association between the types of depression and mental HRQoL (p for interaction = 0.005). Melancholic type of depressive symptoms was associated with higher mental HRQoL compared to the non-melancholic symptoms among individuals with low hePRS-IR (adjusted mean 4.1, 95% CI 0.7-7.4, p = 0.018). However, no such difference was evident in moderate or high hePRS-IR groups as higher hePRS-IR was associated with lower mental HRQoL (B = - 3.4, 95% CI - 5.6 to - 1.2) in individuals with melancholic type of depressive symptoms. No direct associations were detected between the ePRS-IRs and type of depressive symptoms or HRQoL. Variations in the glucose-insulin metabolism can lower HRQoL in individuals with melancholic depressive symptoms.

Phospholipase Cγ2 regulates endocannabinoid and eicosanoid networks in innate immune cells.

Human genetic studies have pointed to a prominent role for innate immunity and lipid pathways in immunological and neurodegenerative disorders. Our understanding of the composition and function of immunomodulatory lipid networks in innate immune cells, however, remains incomplete. Here, we show that phospholipase Cγ2 (PLCγ2 or PLCG2)-mutations in which are associated with autoinflammatory disorders and Alzheimer's disease-serves as a principal source of diacylglycerol (DAG) pools that are converted into a cascade of bioactive endocannabinoid and eicosanoid lipids by DAG lipase (DAGL) and monoacylglycerol lipase (MGLL) enzymes in innate immune cells. We show that this lipid network is tonically stimulated by disease-relevant human mutations in PLCγ2, as well as Fc receptor activation in primary human and mouse macrophages. Genetic disruption of PLCγ2 in mouse microglia suppressed DAGL/MGLL-mediated endocannabinoid-eicosanoid cross-talk and also caused widespread transcriptional and proteomic changes, including the reorganization of immune-relevant lipid pathways reflected in reductions in DAGLB and elevations in PLA2G4A. Despite these changes, Plcg2-/- mice showed generally normal proinflammatory cytokine and chemokine responses to lipopolysaccharide treatment, instead displaying a more restricted deficit in microglial activation that included impairments in prostaglandin production and CD68 expression. Our findings enhance the understanding of PLCγ2 function in innate immune cells, delineating a role in cross-talk with endocannabinoid/eicosanoid pathways and modulation of subsets of cellular responses to inflammatory stimuli.

Recombinant expression, purification, and characterization of full-length human BST-2 from Escherichia coli.

HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.

Eubacterium rectale Attenuates HSV-1 Induced Systemic Inflammation in Mice by Inhibiting CD83.

The purpose of this study was to determine whether administration of the microorganism Eubacterium rectale (E. rectale) could regulate dendritic cell (DC) activation and systemic inflammation in herpes simplex virus type 1-induced Behçet's disease (BD). E. rectale, butyrate-producing bacteria, was administered to BD mice. Peripheral blood leukocytes (PBL) and lymph node cells were isolated and analyzed by flow cytometry. 16S rRNA metagenomic analysis was performed in the feces of mice to determine the differences in the composition of the microbial population between normal and BD mice. Serum cytokine levels were measured by enzyme-linked immunosorbent assay. The frequency of DC activation marker CD83 positive cells was significantly increased in PBL of BD mice. Frequencies of CD83+ cells were also significantly increased in patients with active BD. 16S rRNA metagenomic analysis revealed different gut microbiota composition between normal and BD mice. The administration of E. rectale to BD mice reduced the frequency of CD83+ cells and significantly increased the frequency of NK1.1+ cells with the improvement of symptoms. The co-administration of colchicine and E. rectale also significantly reduced the frequency of CD83+ cells. Differences in gut microbiota were observed between normal mice and BD mice, and the administration of E. rectale downregulated the frequency of CD83, which was associated with BD deterioration. These data indicate that E. rectale could be a new therapeutic adjuvant for BD management.

Expression of Immune Checkpoint Receptors in Placentae With Infectious and Non-Infectious Chronic Villitis.

Pregnancy is an immunological paradox whereby maternal immunity accepts a genetically unique fetus (or fetuses), while maintaining protective innate and adaptive responses to infectious pathogens. This close contact between the genetically diverse mother and fetus requires numerous mechanisms of immune tolerance initiated by trophoblast cell signals. However, in a placental condition known as villitis of unknown etiology (VUE), there appears to be a breakdown in this tolerance allowing maternal cytotoxic T-cells to traffic into the placenta to destroy fetal villi. VUE is associated with several gestational complications and an increased risk of recurrence in a subsequent pregnancy, making it a significant obstetrical diagnosis. The cause of VUE remains unclear, but dysfunctional signaling through immune checkpoint pathways, which have a critical role in blunting immune responses, may play an important role. Therefore, using placental tissue from normal pregnancy (n=8), VUE (n=8) and cytomegalovirus (CMV) infected placentae (n=4), we aimed to identify differences in programmed cell death 1 (PD-1), programmed death ligand-1 (PD-L1), LAG3 and CTLA4 expression between these etiologies by immunohistochemistry (IHC). Results demonstrated significantly lower expression of PD-L1 on trophoblast cells from VUE placentae compared to control and CMV infection. Additionally, we observed significantly higher counts of PD-1+ (>100 cells/image) and LAG3+ (0-120 cells/image) cells infiltrating into the villi during VUE compared to infection and control. Minimal CTLA4 staining was observed in all placentae, with only a few Hofbauer cells staining positive. Together, this suggests that a loss of tolerance through immune checkpoint signaling may be an important mechanism leading to the activation and trafficking of maternal cells into fetal villi during VUE. Further mechanistic studies are warranted to understand possible allograft rejection more clearly and in developing effective strategies to prevent this condition from occurring in utero.

Characterisation of immune checkpoints in Richter syndrome identifies LAG3 as a potential therapeutic target.

Richter syndrome (RS), an aggressive lymphoma occurring in the context of chronic lymphocytic leukaemia/small lymphocytic lymphoma, is associated with poor prognosis when treated with conventional immunochemotherapy, therefore, improved treatments are required. Immune checkpoint blockade has shown efficacy in some B-cell malignancies and modest responses in early clinical trials for RS. We investigated the immune checkpoint profile of RS as a basis to inform rational therapeutic investigations in RS. Formalin-fixed, paraffin-embedded biopsies of RS (n = 19), de novo diffuse large B-cell lymphoma (DLBCL; n = 58), transformed indolent lymphomas (follicular [tFL], n = 16; marginal zone [tMZL], n = 24) and non-transformed small lymphocytic lymphoma (SLL; n = 15) underwent gene expression profiling using the NanoString Human Immunology panel. Copy number assessment was performed using next-generation sequencing. Immunohistochemistry (IHC) for LAG3 and PD-1 was performed. LAG3 gene expression was higher in RS compared to DLBCL (P = 0·0002, log2FC 1·96), tFL (P < 0·0001, log2FC 2·61), tMZL (P = 0·0004, log2FC 1·79) and SLL (P = 0·0057, log2FC 1·45). LAG3 gene expression correlated with the gene expression of human leukocyte antigen Class I and II, and related immune genes and immune checkpoints. IHC revealed LAG3 protein expression on both malignant RS cells and tumour-infiltrating lymphocytes. Our findings support the investigation of LAG3 inhibition to enhance anti-tumour responses in RS.

Caspase-1-Inhibitor AC-YVAD-CMK Inhibits Pyroptosis and Ameliorates Acute Kidney Injury in a Model of Sepsis.

OBJECTIVE: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily. METHODS: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) (n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), TNF-α, and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues. RESULTS: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level (P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls (P < 0.05). CONCLUSIONS: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis.

Tumor-Infiltrating B Lymphocyte Profiling Identifies IgG-Biased, Clonally Expanded Prognostic Phenotypes in Triple-Negative Breast Cancer.

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20+CD27+IgD- isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.

Anti-CD47 antibody administration via cisterna magna in proper dosage can reduce perihematomal cell death following intracerebral hemorrhage in rats.

OBJECTIVE: The secondary injury caused by RBC autolysis after intracerebral hemorrhage (ICH) can be reduced by increasing the efficiency of microglia (MG)/macrophages (Mø) phagocytizing red blood cells (RBCs). CD47 is an important regulator of MG/Mø phagocytosis. This study aims to clarify whether anti-CD47 antibody administrated into the cisterna magna after ICH can transfer to the hematoma site, promote MG/Mø gathering to phagocytize RBCs and ultimately reduce cell death. METHODS: Forty male Wistar rats were divided into sham, ICH, low-dosage (group A, 0.3 µg), medium-dosage (group B, 0.9 µg) and high-dosage (group C, 1.8 µg) anti-CD47 antibody groups. For the rats in group A, B and C, anti-CD47 antibody solution was administrated into the cisterna magna at 10 min after ICH. Brain tissue was harvested 3 days after the operation. Western blotting was performed to detect the expression of Caspase-3 and Bcl-2. Immunofluorescence was performed to detect the CD68 expression. TUNEL was performed to detect the cell death. RESULTS: The hematoma of the ICH rats was located in the basal ganglia, with a good homogeneity of hematoma volume. Low-dosage anti-CD47 antibody in group A had no effects on the perihematomal CD68 (P = 0.338), Caspase-3 (P = 0.769), Bcl-2 (P = 0.176) expression and cell death (P = 0.698), compared with the ICH group. CD68 and Bcl-2 expression increased and Caspase-3 expression decreased significantly in group B (P < 0.001 for all) and group C (P < 0.001 for all). The increase of CD68 expression in group C was greater than that in group B (P < 0.01) by a large margin, while there was no difference for Bcl-2 (P = 0.908) and Caspase-3 (P = 0.913) expression between the 2 groups. Compared with the ICH group, medium-dosage of anti-CD47 antibody in group B significantly reduced the number of TUNEL-positive cells (P < 0.005), but not for group C (P = 0.311). CONCLUSION: The results suggested that anti-CD47 antibody administration into the cisterna magna in proper dosage (0.9 µg) can effectively reach the hematoma, induce more MG/Møs to gather around the hematoma, and reduce cell death in perihematomal brain tissue. The results of this study has provided a basic theory for improving the efficiency of MG/Mø phagocytizing RBCs and hematoma clearance after ICH by administrating anti-CD47 antibody via the cisterna magna.

Programmed death ligand 1 (PD-L1) plays a vital part in DC tolerogenicity induced by IFN-γ.

Interferon-γ (IFN-γ) is the sole representative of type II IFNs, with well recognized role in numerous inflammatory processes. Lately, its significant pleiotropic nature has been recognized in many scenarios, where IFN-γ contributes to maintenance or induction of tolerogenic responses in context of various immune cell types. In this manuscript we demonstrate, that IFN-γ-mediated induction of programmed death ligand 1 (PD-L1) on human monocyte-derived dendritic cells (DCs) represents an important tolerogenic aspect in immunological network of type II IFNs. When fully differentiated, immature DCs were treated with increasing concentrations of IFN-γ there was no sign of maturation, as revealed by CD80, CD83 and CD86 expression. In terms of co-stimulatory receptor response, we did observe a dose-dependent increase in CD40 expression. Phenotypic analysis of inhibitory molecules revealed that PD-L1 expression is particularly sensitive to IFN-γ, as its expression can be induced almost 10-fold in comparison to non-treated DCs. Functional analysis of such PD-L1high DCs revealed significant immunosuppressive properties in a mixed lymphocyte reaction with whole or memory CD4+ T cells. When IFN-γ treated DCs were co-cultured with naive CD4+CD45RA+ T cells, they induced an increased percentage of CD4+CD25+CD127-FoxP3+ Tregs. Inhibition of PD-1/PD-L1 axis using neutralizing anti-PD-L1 mAbs, reversed the immunosuppressive effect of IFN-γ-treated DCs to suppress CD4+ T cell proliferation and to induce Tregs. In summary, our findings demonstrate the importance of IFN-γ-mediated tolerogenic effects, exerted on DCs by inducing increased expression of PD-L1, which enhances their regulatory function.

The differentiation of new human CD303+ Plasmacytoid dendritic cell subpopulations expressing CD205 and/or CD103 regulated by Non-Small-Cell lung cancer cells.

CD303+ plasmacytoid dendritic cells (pDCs) play an important role in the induction of immune tolerance and antitumor immunity. Here, we focused on the effect of NSCLC cells on the development of CD303+ pDC subsets expressing CD205 and/or CD103. The NSCLC cell line H1299 and primary NSCLC cells were incubated with DCs. The protein expression of costimulatory molecules on CD303+ pDCs, the production of pro-inflammatory and anti-inflammatory cytokines by CD303+ pDCs and the development of CD303+ pDC subsets were detected by using flow cytometry. Coculture with NSCLC cells modulates the protein expression of CD86 and HLA-DR on CD303+ pDCs. Moreover, NSCLC cells suppressed the production of IL-12 and IL-23 but facilitated the secretion of IL-27 and TGF-ß by CD303+ pDCs. There were new CD303+ pDC subsets expressing CD205 and/or CD103 in healthy donors and NSCLC patients: CD303+CD205+CD103+, CD303+CD205+CD103-, CD303+CD205-CD103+ and CD303+CD205-CD103- pDCs. NSCLC cells modulated the differentiation of CD303+ pDC subpopulations by regulating the protein expression of CD205 and/or CD103 on CD303+ pDCs. NSCLC cells may regulate the immune functions of CD303+ pDCs by modulating the expression of costimulatory molecules on DCs and the production of pro-inflammatory/anti-inflammatory cytokines by DCs. NSCLC cells also regulate the development of CD303+ pDC subsets expressing CD205 and/or CD103. These outcomes may reveal a new cellular mechanism leading to the NSCLC-induced immune-suppressive microenvironment.

Loss of CD96 Expression as a Marker of HIV-Specific CD8+ T-Cell Differentiation and Dysfunction.

Persistent immune activation and inflammation in people living with HIV (PLWH) are associated with immunosenescence, premature aging and increased risk of non-AIDS comorbidities, with the underlying mechanisms not fully understood. In this study, we show that downregulation of the T-cell immunoglobulin receptor CD96 on CD8+ T cells from PLWH is associated with decreased expression of the co-stimulatory receptors CD27 and CD28, higher expression of the senescence marker CD57 and accumulation of a terminally differentiated T-cell memory phenotype. In addition, we show that CD96-low CD8+ T-cells display lower proliferative potential compared to their CD96-high counterparts and that loss of CD96 expression by HIV-specific CD8+ T-cells is associated with a suboptimal response to HIV antigens. In conclusion, our results suggest that CD96 marks CD8+ T-cells with competent responses to HIV and the loss of its expression might be used as a biomarker for CD8+ T-cell senescence and dysfunction in PLWH.

Phenotypic and Functional Consequences of PLT Binding to Monocytes and Its Association with Clinical Features in SLE.

Platelets (PLTs) can modulate the immune system through the release of soluble mediators or through interaction with immune cells. Monocytes are the main immune cells that bind with PLTs, and this interaction is increased in several inflammatory and autoimmune conditions, including systemic lupus erythematosus (SLE). Our aim was to characterize the phenotypic and functional consequences of PLT binding to monocytes in healthy donors (HD) and in SLE and to relate it to the pathogenesis of SLE. We analyzed the phenotypic and functional features of monocytes with non-activated and activated bound PLTs by flow cytometry. We observed that monocytes with bound PLTs and especially those with activated PLTs have an up-regulated HLA-DR, CD86, CD54, CD16 and CD64 expression. Monocytes with bound PLTs also have an increased capacity for phagocytosis, though not for efferocytosis. In addition, monocytes with bound PLTs have increased IL-10, but not TNF-α, secretion. The altered phenotypic and functional features are comparable in SLE and HD monocytes and in bound PLTs. However, the percentages of monocytes with bound PLTs are significantly higher in SLE patients and are associated with undetectable levels of anti-dsDNA antibodies and hematuria, and with normal C3 and albumin/creatinine levels. Our results suggest that PLTs have a modulatory influence on monocytes and that this effect may be highlighted by an increased binding of PLTs to monocytes in autoimmune conditions.

Single-Cell RNA Sequencing Reveals Tissue Compartment-Specific Plasticity of Mycosis Fungoides Tumor Cells.

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (TRM, CD69+CD27-NR4A1+RGS1+AHR+ ). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (KLF2+TCF7+S1PR1+SELL+CCR7+ ), while retaining tissue-homing receptors (CLA, CCR10). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators (CCL13, CCL17, CCL22). Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by TIGIT+CTLA4+CSF2+TNFSF14+ tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the TRM-like phenotype enables long-term skin residence of MF cells. Their switch to a TCM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.

Single-cell Profiles and Prognostic Impact of Tumor-Infiltrating Lymphocytes Coexpressing CD39, CD103, and PD-1 in Ovarian Cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8+ TIL in various cancers. We evaluated the phenotype, clonality, and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches. EXPERIMENTAL DESIGN: Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens. RESULTS: Coexpression of CD39, CD103, and PD-1 ("triple-positive" phenotype) demarcated subsets of CD8+ TIL and CD4+ regulatory T cells (Treg) with a highly activated/exhausted phenotype. Triple-positive CD8+ TIL exhibited reduced T-cell receptor (TCR) diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8+ effector cells relative to their CD4+ Treg counterparts. CONCLUSIONS: Coexpression of CD39, CD103, and PD-1 demarcates highly activated CD8+ and CD4+ TIL with inferred roles in cytolytic, humoral, and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39, and TIGIT.