Effector Responses Research Articles

Leukemia inhibitory factor (LIF) receptor amplifies pathogenic activation of fibroblasts in lung fibrosis

Fibrosis drives end-organ damage in many diseases. However, clinical trials targeting individual upstream activators of fibroblasts, such as TGFβ, have largely failed. Here, we target the leukemia inhibitory factor receptor (LIFR) as an "autocrine master amplifier" of multiple upstream activators of lung fibroblasts. In idiopathic pulmonary fibrosis (IPF), the most common fibrotic lung disease, we found that lung myofibroblasts had high LIF expression, and the fibroblasts in fibroblastic foci coexpressed LIF and LIFR. In IPF, fibroblastic foci are the "leading edge" of fibrosis and a key site of disease pathogenesis. TGFβ1, one of the principal drivers of fibrosis, up-regulated LIF expression in IPF fibroblasts. We found that TGFβ1, IL-4, and IL-13 stimulations of fibroblasts require the LIF-LIFR axis to evoke a strong fibrogenic effector response in fibroblasts. In vitro antibody blockade of LIFR on IPF lung fibroblasts reduced the induction of profibrotic genes after TGFβ1 stimulation. Silencing LIF and LIFR reduced profibrotic fibroblast activation following TGFβ1, IL-4, and IL-13 stimulations. We also demonstrated that LIFR amplified profibrotic stimuli in precision-cut lung slices from IPF patients. These LIFR signals were transduced via JAK2, and STAT1 in IPF lung fibroblasts. Together, we find that LIFR drives an autocrine circuit that amplifies and sustains pathogenic activation of IPF fibroblasts. Targeting a single, downstream master amplifier on fibroblasts, like LIFR, is an alternative therapeutic strategy that simultaneously attenuates the profibrotic effects of multiple upstream stimuli.

Seroprevalence study reveals pertussis underreporting in Brazil and calls for adolescent/young adult boosting: mouse model demonstrates immunity restoration.

Pertussis continues to pose a significant threat despite the availability of effective vaccines. The challenge lies in the vulnerability of infants who have not yet completed their vaccination schedule and in adolescents and adults becoming potential disease carriers. We evaluated the seroprevalence of pertussis immunity in a cohort of 1,500 healthy Brazilian volunteers. Next, we explored the potential restoration of waning pertussis immunity by administering booster doses of wP, aP or Plow (an economically viable and low reactogenic vaccine in development at Butantan) using a mouse model. The mean anti-PT IgG levels in the Brazilian volunteers was 39.4 IU/mL. Notably, individuals ≤ 19 years exhibited higher IgG values compared to older age groups (≥ 20 y). Overall, 8.4% of the samples displayed indications of recent or current contact/infection, with IgG levels surpassing 120 IU/mL, particularly in the 15-19 years age group. IgM values were also increased in the 10-19 years age group. Potential recovery of pre-existing but waning immunity investigated in mice, showed that boosting with wP induced higher antibody titers than aP or Plow. Notably, aP and Plow boosts prompted superior effector and memory cell responses from both B and T cells. Upon challenge with B. pertussis, aP or Plow boost provided greater protection as compared to wP. Pertussis appears to circulate predominantly among adolescents and young adults. Insights from the mouse model indicate that immunity can be restored with booster doses. Boosting immunity in non-targeted groups could prevent the dissemination of pertussis to infants.

Cholesterol promotes IFNG mRNA expression in CD4+ effector/memory cells by SGK1 activation.

IFNγ-secreting T cells are central for the maintenance of immune surveillance within the central nervous system (CNS). It was previously reported in healthy donors that the T-cell environment in the CNS induces distinct signatures related to cytotoxic capacity, CNS trafficking, tissue adaptation, and lipid homeostasis. These findings suggested that the CNS milieu consisting predominantly of lipids mediated the metabolic conditions leading to IFNγ-secreting brain CD4 T cells. Here, we demonstrate that the supplementation of CD4+CD45RO+CXCR3+ cells with cholesterol modulates their function and increases IFNG expression. The heightened IFNG expression was mediated by the activation of the serum/glucocorticoid-regulated kinase (SGK1). Inhibition of SGK1 by a specific enzymatic inhibitor significantly reduces the expression of IFNG Our results confirm the crucial role of lipids in maintaining T-cell homeostasis and demonstrate a putative role of environmental factors to induce effector responses in CD4+ effector/memory cells. These findings offer potential avenues for further research targeting lipid pathways to modulate inflammatory conditions.

IMMU-38. LEVERAGING INNATE IMMUNE SENSORS TO GENERATE DURABLE ANTI-GLIOMA ADAPTIVE IMMUNE RESPONSES

Abstract Glioblastoma harbors a tumor microenvironment (TME) that is mostly devoid of T cell effector infiltration but is dominated by immune suppressive microglia, macrophages, and myeloid-derived suppresser cells. The innate immune cells in the TME have impaired phagocytosis and antigen presentation and are inadequate for triggering the subsequent immune activating signals needed for anti-tumor effector responses. Phagocytic activity can also be blocked by the upregulation of CD47 on the tumor cells. In the immune checkpoint resistant Qki-/- Pten-/- P53-/- (QPP) glioma model, intratumoral treatment with the synthetic cyclic di-nucleotide STING agonist IACS-8803 (8803) induces global immunological reprogramming and long-term survival resistant to tumor rechallenge. More specifically, in the infiltrating myeloid stroma 8803 increases costimulatory protein CD86 while reducing the expression of suppressive markers CD163 and CD206. Conventional type 1 dendritic cells are increased in both the TME (p<0.05) and the draining cervical lymph node (p<0.01). Intratumoral infiltration of both CD8 T (p<0.05) and NK (p<0.001) cells increases. However, only T cells, but not NK cells, are necessary for 8803 therapeutic activity when assessed with in vivo NK cell depletion studies and treatment in the RAG-/- background. STING activation increases microglial phagocytic capacity, but also induces a compensatory increase in SIRPα expression (p<0.001) – the ligand for CD47. 8803 synergizes with anti-CD47 blockade to enhance microglia phagocytosis of QPP glioblastoma cells in vitro. Finally, the combination of 8803 and anti-CD47 synergizes to extend both median survival and the fraction of long-term survivors in orthotopic QPP-bearing mice (QPP8v; vehicle control MS=39d, anti-CD47 MS=40d, 8803 MS=67d 20% long-term survival, 8803 + anti-CD47 MS=107.5d 40% long-term survival). These data demonstrate that 8803 induces proinflammatory conversion of the glioblastoma TME, generates T cell-dependent survival benefits in QPP-bearing mice, and synergizes with anti-CD47 blockade to enhance microglia phagocytosis of glioblastoma cells and extend survival in QPP-bearing mice.

Targeting heterogeneous tumor microenvironments in pancreatic cancer mouse models of metastasis by TGF-β depletion.

The dual tumor-suppressive and -promoting functions of TGF-β signaling has made its targeting challenging. We examined the effects of TGF-β depletion by AVID200/BMS-986416 (TGF-β-TRAP), a TGF-β ligand trap, on the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) murine models with different organ-specific metastasis. Our study demonstrated that TGF-β-TRAP potentiates the efficacy of anti-programmed cell death 1 (anti-PD-1) in a PDAC orthotopic murine model with liver metastasis tropism, significantly reducing liver metastases. We further demonstrated the heterogeneous response of cytotoxic effector T cells to combination TGF-β-TRAP and anti-PD-1 treatment across several tumor models. Single-nuclear RNA sequencing suggested that TGF-β-TRAP modulates cancer-associated fibroblast (CAF) heterogeneity and suppresses neutrophil degranulation and CD4+ T cell response to neutrophil degranulation. Ligand-receptor analysis indicated that TGF-β-TRAP may modulate the CCL5/CCR5 axis as well as costimulatory and checkpoint signaling from CAFs and myeloid cells. Notably, the most highly expressed ligands of CCR5 shifted from the immunosuppressive CCL5 to CCL7 and CCL8, which may mediate the immune agonist activity of CCR5 following TGF-β-TRAP and anti-PD-1 combination treatment. This study suggested that TGF-β depletion modulates CAF heterogeneity and potentially reprograms CAFs and myeloid cells into antitumor immune agonists in PDAC, supporting the validation of such effects in human specimens.

Spatial multiplex analysis of lung cancer reveals that regulatory T cells attenuate KRAS-G12C inhibitor-induced immune responses.

Kirsten rat sarcoma virus (KRAS)-G12C inhibition causes remodeling of the lung tumor immune microenvironment and synergistic responses to anti-PD-1 treatment, but only in T cell infiltrated tumors. To investigate mechanisms that restrain combination immunotherapy sensitivity in immune-excluded tumors, we used imaging mass cytometry to explore cellular distribution in an immune-evasive KRAS mutant lung cancer model. Cellular spatial pattern characterization revealed a community where CD4+ and CD8+ T cells and dendritic cells were gathered, suggesting localized T cell activation. KRAS-G12C inhibition led to increased PD-1 expression, proliferation, and cytotoxicity of CD8+ T cells, and CXCL9 expression by dendritic cells, indicating an effector response. However, suppressive regulatory T cells (Tregs) were also found in frequent contact with effector T cells within this community. Lung adenocarcinoma clinical samples showed similar communities. Depleting Tregs led to enhanced tumor control in combination with anti-PD-1 and KRAS-G12C inhibitor. Combining Treg depletion with KRAS inhibition shows therapeutic potential for increasing antitumoral immune responses.

Physiological and pathogenic T cell autoreactivity converge in type 1 diabetes

Autoimmune diseases result from autoantigen-mediated activation of adaptive immunity; intriguingly, autoantigen-specific T cells are also present in healthy donors. An assessment of dynamic changes of this autoreactive repertoire in both health and disease is thus warranted. Here we investigate the physiological versus pathogenic autoreactive processes in the context of Type 1 diabetes (T1D) and one of its landmark autoantigens, glutamic acid decarboxylase 65 (GAD65). Using single cell gene expression profiling and tandem T cell receptor (TCR) sequencing, we find that GAD65-specific true naïve cells are present in both health and disease, with GAD65-specific effector and memory responses showing similar ratios in healthy donors and patients. Deeper assessment of phenotype and TCR repertoire uncover differential features in GAD65-specific TCRs, including lower clonal sizes of healthy donor-derived clonotypes in patients. We thus propose a model whereby physiological autoimmunity against GAD65 is needed during early life, and that alterations of these physiological autoimmune processes in predisposed individuals trigger overt Type 1 diabetes.

Concurrent hypoxia and apoptosis imparts immune programming potential in mesenchymal stem cells: Lesson from acute graft-versus-host-disease model

BackgroundMesenchymal stem cells (MSCs) have emerged as promising candidates for immune modulation in various diseases that are associated with dysregulated immune responses like Graft-versus-Host-Disease (GVHD). MSCs are pleiotropic and the fate of MSCs following administration is a major determinant of their therapeutic efficacy.MethodsHuman MSCs were derived from bone marrow (BM) and Wharton’s Jelly (WJ) and preconditioned through exposure to hypoxia and induction of apoptosis, either sequentially or simultaneously. The immune programming potential of preconditioned MSCs was evaluated by assessing their effects on T cell proliferation, induction of Tregs, programming of effector T-cell towards Th2 phenotype, macrophage polarization in the direct co-culture of MSCs and aGVHD patients-derived PBMNCs. Additionally, efferocytosis of MSCs and relative change in the expression of immunomodulatory soluble factors were examined.ResultsOur study demonstrated that hypoxia preconditioned apoptotic MSCs (BM-MSCs, WJ-MSCs) bear more immune programming ability in a cellular model of acute Graft-versus-Host-Disease (aGVHD). Our findings revealed that WJ-MSCsHYP+APO were superior to BM-MSCsHYP+APO for immune regulation. These induced the differentiation of CD4+T-cell into Tregs, enhanced Th2 effector responses, and simultaneously mitigated Th1- and Th17 responses. Additionally, this approach led to the polarization of M1 macrophages toward an M2 phenotype.ConclusionOur study highlights the potential of WJ-MSCs conditioned with hypoxia and apoptosis concurrently, as a promising therapeutic strategy for aGVHD. It underscores the importance of considering MSC apoptosis in optimizing MSCs-based cellular therapy protocols for enhanced therapeutic efficacy in aGvHD.Graphical

Nitric oxide-producing monocyte-myeloid suppressor cells expand and accumulate in the spleen and mesenteric lymph nodes of Yersinia enterocolitica-infected mice.

Yersinia enterocolitica (Ye) is a Gram-negative bacterium that causes gastrointestinal infections. The myeloid-derived suppressor cells (MDSCs) constitute a cellular population with the capacity of inducing the specific suppression of T cells. Although there is evidence supporting the role of MDSCs in controlling the immune responses in several bacterial infections, its role during Ye infection has not yet been reported. Therefore, the purpose of the present work was to analyze MDSCs after oral Ye infection. C57BL/6 wild-type mice were infected with Ye WAP-314 serotype O:8. The proliferation of splenocytes and mesenteric lymph nodes (MLN) cells was measured as well as the levels of cytokines and nitric oxide (NO) in culture supernatants. The frequency and subsets of MDSCs were analyzed in the intestinal mucosa and spleen by flow cytometry. Furthermore, monocytic-MDSCs (Mo-MDSCs) and polymorphonuclear-MDSCs (PMN-MDSCs) were purified from the spleen of infected mice and their suppressor activity was evaluated in co-cultures with purified T cells. we observed a marked expansion of CD11b+Gr-1+ cells, a phenotype consistent with MDSCs, in the spleen and intestinal mucosa of Ye-infected mice. Interestingly, a robust proliferation of splenocytes and MLN cells was observed only when the MDSCs were depleted or the NO production was blocked. In addition, we determined that only Mo-MDSCs had the ability to suppress T-cell proliferation. Our results highlight a mechanism by which Ye may induce suppression of the immune responses. We suggest that NO-producing Mo-MDSCs expand and accumulate in MLN and spleen of Ye-infected mice. These cells can then suppress the T-cell function without interfering with the anti-bacterial effector response. Instead, these immature myeloid cells may perform an important function in regulating the inflammatory response and protecting affected tissues.

Modelling and investigating memory immune responses in infectious disease. Application to influenza a virus and sars-cov-2 reinfections

Understanding effector and memory immune responses against influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and re-infections is extremely important, given that they are now endemic in the community. The goal of this study is to investigate the role of memory cells and antibodies in the immune responses against IAV and SARS-CoV-2 re-infections. To this end, we adapt a previously-published within-host mathematical model (Sadria & Layton, 2021) for the primary immune response against SARS-CoV-2 infections, by including two types of memory immune cells, i.e., memory CD8+ T-cells and memory B-cells, and by parametrising the new model with values specific to the two viruses. We first investigate the long-term dynamics of the model by identifying the virus-free steady states and studying the conditions that ensure the stability of these states. Then, we investigate the transient dynamics of this in-host model by simulating different viral reinfection times: 20 days, 60 days and 400 days after the first encounter with the pathogen. This allows us to highlight which memory immune components have the greatest impact on the viral elimination depending on the time of reinfection. Our results suggest that memory immune responses have a greater impact in the case of IAV infections compared to SARS-CoV-2 infections. Moreover, we observe that the immune response after a secondary infection is more efficient when the reinfection occurs at a shorter time.

Functional plasticity shapes neutrophil response to Leishmania major infection in susceptible and resistant strains of mice.

Neutrophils rapidly infiltrate sites of infection and possess several microbicidal strategies, such as neutrophil extracellular traps release and phagocytosis. Enhanced neutrophil infiltration is associated with higher susceptibility to Leishmania infection, but neutrophil effector response contribution to this phenotype is uncertain. Here, we show that neutrophils from susceptible BALB/c mice (B/c) produce more NETs in response to Leishmania major than those from resistant C57BL/6 mice (B6), which are more phagocytic. The absence of neutrophil elastase contributes to phagocytosis regulation. Microarray analysis shows enrichment of genes involved in NET formation (mpo, pi3kcg, il1b) in B/c, while B6 shows upregulation of genes involved in phagocytosis and cell death (Arhgap12, casp9, mlkl, FasL). scRNA-seq in L. major-infected B6 showed heterogeneity in the pool of intralesional neutrophils, and we identified the N1 subset as the putative subpopulation involved with phagocytosis. In vivo, imaging validates NET formation in infected B/c ears where NETing neutrophils were mainly uninfected cells. NET digestion in vivo augmented parasite lymphatic drainage. Hence, a balance between NET formation and phagocytosis in neutrophils may contribute to the divergent phenotype observed in these mice.

Targeting HLA-E-overexpressing cancers with a NKG2A/C switch receptor

Human leukocyte antigen (HLA)-E is overexpressed by a large proportion of solid tumors, including malignant glioblastoma, and acts as a major checkpoint for NKG2A+ CD8+ Tcells and natural killer (NK) cells in the tumor microenvironment and circulation. This axis operates alongside PD-L1 to inhibit effector responses by T and NK cells. We engineered a chimeric A/C switch receptor, combining the high HLA-E binding affinity of the NKG2A receptor ectodomain with the activating signaling of the NKG2C receptor endodomain. The cytotoxic function of A/C switch-transduced NK and Tcells was evaluated against tumor cells with varying levels of HLA-E expression. Invivo efficacy was assessed using a xenograft model of glioblastoma. A/C switch-transduced NK and Tcells exhibited superior and specific cytotoxicity against tumor cells with medium to high HLA-E expression. A/C switch-expressing human Tcells demonstrated enhanced anti-tumor function in a glioblastoma xenograft model. The activity of the modified Tcells was governed by an equilibrium between A/C switch levels and HLA-E expression, creating a therapeutic window to minimize on-target, off-tumor toxicities. Normal cells remained insensitive to A/C switch Tcells, even after interferon (IFN)-γ pretreatment to induce HLA-E expression. The A/C switch receptor effectively targets tumor cells expressing high levels of HLA-E, either alone or in combination with other engineered specificities, to overcome the suppressive NKG2A/HLA-E checkpoint. This approach offers a promising therapeutic strategy with a favorable safety profile for targeting HLA-E-overexpressing tumors. This work was funded by The Research Council of Norway, the Norwegian Cancer Society, and the National Cancer Institute.

Local receptor-interacting protein kinase 2 inhibition mitigates house dust mite-induced asthma.

House dust mite is the most frequent trigger of allergic asthma, with innate and adaptive immune mechanisms playing critical roles in outcomes. We recently identified the nucleotide-binding oligomerisation domain 1 (NOD1)/receptor-interacting serine/threonine protein kinase 2 (RIPK2) signalling pathway as a relevant contributor to murine house dust mite-induced asthma. This study aimed to evaluate the effectiveness of a pharmacological RIPK2 inhibitor administered locally as a preventive and therapeutic approach using a house dust mite-induced asthma model in wild-type and humanised NOD1 mice harbouring an asthma-associated risk allele, and its relevance using air-liquid interface epithelial cultures from asthma patients. A RIPK2 inhibitor was administered intranasally either preventively or therapeutically in a murine house dust mite-induced asthma model. Airway hyperresponsiveness, bronchoalveolar lavage composition, cytokine/chemokine expression and mucus production were evaluated, as well as the effect of the inhibitor on precision-cut lung slices. Furthermore, the inhibitor was tested on air-liquid interface epithelial cultures from asthma patients and controls. While local preventive administration of the RIPK2 inhibitor reduced airway hyperresponsiveness, eosinophilia, mucus production, T-helper type 2 cytokines and interleukin 33 (IL-33) in wild-type mice, its therapeutic administration failed to reduce the above parameters, except IL-33. By contrast, therapeutic RIPK2 inhibition mitigated all asthma features in humanised NOD1 mice. Results in precision-cut lung slices emphasised an early role of thymic stromal lymphopoietin and IL-33 in the NOD1-dependent response to house dust mite, and a late effect of NOD1 signalling on IL-13 effector response. RIPK2 inhibitor downregulated thymic stromal lymphopoietin and chemokines in house dust mite-stimulated epithelial cultures from asthma patients. These data support that local interference of the NOD1 signalling pathway through RIPK2 inhibition may represent a new therapeutic approach in house dust mite-induced asthma.

Small extracellular vesicles derived from synovial fibroblasts contain distinct miRNA profiles and contribute to chondrocyte damage in osteoarthritis

BackgroundSmall extracellular vesicles (sEV) derived from synovial fibroblasts (SF) represent a novel molecular mechanism regulating cartilage erosion in osteoarthritis (OA). However, a comprehensive evaluation using disease relevant cells has not been undertaken. The aim of this study was to isolate and characterise sEV from OA SF and to look at their ability to regulate OA chondrocyte effector responses relevant to disease. Profiling of micro (mi) RNA signatures in sEV and parental OA SF cells was performed.MethodsSF and chondrocytes were isolated from OA synovial membrane and cartilage respectively (n = 9). sEV were isolated from OA SF (± IL-1β) conditioned media by ultracentrifugation and characterised using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Particle size was confirmed by nanoparticle tracking analysis (NTA). sEV regulation of OA chondrocyte and cartilage effector response was evaluated using qPCR, ELISA and sulphated glycosaminoglycan assay (sGAG). RNA-sequencing was used to establish miRNA signatures in isolated sEV from OA SF.ResultsOA SF derived sEV were readily taken up by OA chondrocytes, with increased expression of the catabolic gene MMP 13 (p < 0.01) and decreased expression of the anabolic genes aggrecan and COL2A1 (p < 0.01) observed. Treatment with sEV derived from IL-1β stimulated OA SF significantly decreased expression of aggrecan and COL2A1 (p < 0.001) and increased SOX 9 gene expression (p < 0.05). OA chondrocytes cultured with sEV from either non-stimulated or IL-1β treated OA SF, resulted in a significant increase in the secretion of IL-6, IL-8 and MMP-3 (p < 0.01). Cartilage explants cultured with sEV from SF (± IL-1β) had a significant increase in the release of sGAG (p < 0.01). miRNA signatures differed between parental SF cells and isolated sEV. The recently identified osteoclastogenic regulator miR182, along with miR4472-2, miR1302-3, miR6720, miR6087 and miR4532 were enriched in sEV compared to parental cells, p < 0.01. Signatures were similar in sEVs derived from non-stimulated or IL-1β stimulated SF.ConclusionsOA SF sEV regulate chondrocyte inflammatory and remodelling responses. OA SF sEV have unique signatures compared to parental cells which do not alter with IL-1β stimulation. This study provides insight into a novel regulatory mechanism within the OA joint which could inform future targeted therapy.

Blockade of the CCR3 receptor reduces neutrophil recruitment to the lung during acute inflammation.

Neutrophils represent one of the host's first lines of defense against invading pathogens. However, an aberrant activation can cause damage to the host. In the case of respiratory infections with viral or bacterial pathogens, one of the most common complications is the development of acute respiratory distress syndrome, in which neutrophil infiltration into the lung is a hallmark. Neutrophils gain expression of chemokine receptors under inflammatory conditions, and their activation can amplify the neutrophil responses. Earlier studies showed that neutrophils recruited to the lung mucosa during bacterial infection upregulate expression of CCR3 and ex vivo stimulation of CCR3 results in an increased neutrophil activation. Therefore, the modulation of effector functions or migration of neutrophils to target sites through chemokine receptors constitutes an opportunity for pharmacological intervention. We aimed to determine whether the blockade of the CCR3 using the specific antagonist SB-328437 reduces neutrophil recruitment and inflammation in the lung in the lipopolysaccharide (LPS)-induced lung injury model and influenza infection in mice. We found that neutrophils acquire CCR3 expression in the lung alveolar space. The intraperitoneal administration of SB-328437 reduced neutrophil recruitment to the lung alveolar space and reduced tissue damage in both the LPS-induced lung injury model and influenza infection. Moreover, treatment with SB-328437 reduced the percentage of neutrophils producing TNFα and neutrophil activation in the alveolar space. Together, these data suggest that CCR3 blockade might be a pharmacological strategy to prevent the aberrant neutrophil activation that results detrimental for the host but preserves sufficient effector response to control the pathogen.

Breast cancer cell derived exosomes reduces glycolysis of activated CD8 + T cells in a AKT-mTOR dependent manner.

Cytotoxic CD8+ T cells plays a pivotal role in the adaptive immune system to protect the organism against infections and cancer. During activation and response, T cells undergo a metabolic reprogramming that involves various metabolic pathways, with a predominant reliance on glycolysis to meet their increased energy demands and enhanced effector response. Recently, extracellular vesicles (EVs) known as exosomes have been recognized as crucial signaling mediators in regulating the tumor microenvironment (TME). Recent reports indicates that exosomes may transfer biologically functional molecules to the recipient cells, thereby facilitate cancer progression, angiogenesis, metastasis, drug resistance, and immunosuppression by reprogramming the metabolism of cancer cells. This study sought to enlighten possible involvement of cancer-derived exosomes in CD8 + T cell glucose metabolism and discover a regulated signalome as a mechanism of action. We observed reduction in glucose metabolism due to downregulation of AKT/mTOR signalome in activated CD8 + T cells after cancer derived exosome exposure. In-vivo murine breast tumor studies showed better tumor control and antitumor CD8 + T cell glycolysis and effector response after abrogation of exosome release from breast cancer cells. Summarizing, the present study establishes an immune evasion mechanism of breast cancer cell secreted exosomes that will act as a foundation for future precision cancer therapeutics.

ACLY and ACSS2 link nutrient-dependent chromatin accessibility to CD8 T cell effector responses.

Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

Hyperthermia and Exertional Heatstroke During Running, Cycling, Open Water Swimming, and Triathlon Events.

Few previous epidemiological studies, sports medicine position statements, and expert panel consensus reports have evaluated the similarities and differences of hyperthermia and exertional heatstroke (EHS) during endurance running, cycling, open water swimming, and triathlon competitions. Accordingly, we conducted manual online searches of the PubMed and Google Scholar databases using pre-defined inclusion criteria. The initial manual screenings of 1192 article titles and abstracts, and subsequent reviews of full-length pdf versions identified 80 articles that were acceptable for inclusion. These articles indicated that event medical teams recognized hyperthermia and EHS in the majority of running and triathlon field studies (range, 58.8 to 85.7%), whereas few reports of hyperthermia and EHS appeared in cycling and open water swimming field studies (range, 0 to 20%). Sports medicine position statements and consensus reports also exhibited these event-specific differences. Thus, we proposed mechanisms that involved physiological effector responses (sweating, increased skin blood flow) and biophysical heat transfer to the environment (evaporation, convection, radiation, and conduction). We anticipate that the above information will help race directors to distribute pre-race safety advice to athletes and will assist medical directors to better allocate medical resources (eg, staff number and skill sets, medical equipment) and optimize the management of hyperthermia and EHS.

NK cell line modified to express a potent, DR5 specific variant of TRAIL, show enhanced cytotoxicity in ovarian cancer models

ObjectiveOvarian cancer is a lethal gynaecological malignancy with unsatisfactory 5 year survival rates of 30–50 %. Cell immunotherapy is a promising new cancer treatment where immune cells, such as Natural Killer (NK) cells, are administered to enable the patient to fight cancer through direct cytotoxicity. NK cells orchestrate an adaptive immune response by enabling the release of tumour antigens. NK cell cytotoxicity and effector responses are largely driven by TRAIL engagement. In this study we investigated the cytotoxic potential of a human NK cell line that were modified to express a potent DR5 specific TRAIL variant. We hypothesised that this modification would enhance NK cell cytotoxicity against TRAIL sensitive and resistant ovarian cancer cell lines in vitro. MethodsKHYG-1 human NK cells were modified with a TRAIL variant targeting DR5 (TRAILv-KHYG-1). Human ovarian cancer cell lines, OVCAR-3 and SKOV-3, were cultured with modified or non-modified NK cells at different effector:target (E:T) ratios for 4 or 16 h. Apoptosis was assessed by Annexin-APC and 7-AAD and measured using flow cytometry. Apoptotic cells were defined as annexin V 7-AAD double positive. Cytokine expression was measured by multiplex ELISA, and analysed by flow cytometry. ResultsModified and non-modified NK cells significantly reduced OVCAR-3 cell viability as compared to OVCAR-3 cells that were cultured alone after 4 and 16 h treatment. OVCAR-3 cell viability was reduced after treatment with 1:1 E:T ratio with TRAILv-KHYG-1 cells after 16 h. On the contrary, neither NK cell line had any effect of SKOV-3 cell viability despite SKOV-3 cells having more DR5 surface expression compared to OVCAR-3 cells. ConclusionsTRAILv-KHYG-1 cells significantly reduced OVCAR-3 cell viability as compared to non-modified NK cells. However, no significant reduction in viability was observed when SKOV-3 cell were cultured with either NK cells, despite having more DR5 surface expression compared to OVCAR-3 cells. These data indicate that mechanisms other than DR5 expression drive TRAIL resistance in ovarian cancer.

Metformin Restrains the Proliferation of CD4+ T Lymphocytes by Inducing Cell Cycle Arrest in Normo- and Hyperglycemic Conditions.

CD4+ T lymphocytes play a key role in the modulation of the immune response by orchestrating both effector and regulatory functions. The effect of metformin on the immunometabolism of CD4+ T lymphocytes has been scarcely studied, and its impact under high glucose conditions, particularly concerning effector responses and glucose metabolism, remains unknown. This study aims to evaluate the effect of metformin on the modulation of the effector functions and glucose metabolism of CD4+ T lymphocytes under normo- and hyperglycemic conditions. CD4+ T lymphocytes, obtained from peripheral blood from healthy volunteers, were anti-CD3/CD28-activated and cultured for 4 days with three concentrations of metformin (0.1 mM, 1 mM, and 5 mM) under normoglycemic (5.5 mM) and hyperglycemic (25 mM) conditions. Effector functions such as proliferation, cell count, cell cycle analysis, activation markers and cytokine secretion were analyzed by flow cytometry. Glucose uptake was determined using the 2-NBDG assay, and levels of glucose, lactate, and phosphofructokinase (PFK) activity were assessed by colorimetric assays. Metformin at 5 mM restrained the cell counts and proliferation of CD4+ T lymphocytes by arresting the cell cycle in the S/G2 phase at the beginning of the cell culture, without affecting cell activation, cytokine production, and glucose metabolism. In fact, CD69 expression and IL4 secretion by CD4+ T lymphocytes was higher in the presence of 5 mM than the untreated cells in both glucose conditions. Overall, metformin inhibited proliferation through mechanisms associated with cell cycle arrest, leading to an increase in the S/G2 phases at the expense of G1 in activated CD4+ T lymphocytes in normo- and hyperglycemic conditions. Despite the cell cycle arrest, activated CD4+ T lymphocytes remained metabolically, functionally, and phenotypically activated.