PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells.

Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1-4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5-9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2-EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

Rebuilding and rebooting immunity with stem cells.

Advances in modern medicine have enabled a rapid increase in lifespan and, consequently, have highlighted the immune system as a key driver of age-related disease. Immune regeneration therapies present exciting strategies to address age-related diseases by rebooting the host's primary lymphoid tissues or rebuilding the immune system directly via biomaterials or artificial tissue. Here, we identify important, unanswered questions regarding the safety and feasibility of these therapies. Further, we identify key design parameters that should be primary considerations guiding technology design, including timing of application, interaction with the host immune system, and functional characterization of the target patient population.

Hematopoietic stem and progenitor cells improve survival from sepsis by boosting immunomodulatory cells.

New therapeutic strategies to reduce sepsis-related mortality are urgently needed, as sepsis accounts for one in five deaths worldwide. Since hematopoietic stem and progenitor cells (HSPCs) are responsible for producing blood and immune cells, including in response to immunological stress, we explored their potential for treating sepsis. In a mouse model of Group A Streptococcus (GAS)-induced sepsis, severe immunological stress was associated with significant depletion of bone marrow HSPCs and mortality within approximately 5-7 days. We hypothesized that the inflammatory environment of GAS infection drives rapid HSPC differentiation and depletion that can be rescued by infusion of donor HSPCs. Indeed, infusion of 10,000 naïve HSPCs into GAS-infected mice resulted in rapid myelopoiesis and a 50-60% increase in overall survival. Surprisingly, mice receiving donor HSPCs displayed a similar pathogen load compared to untreated mice. Flow cytometric analysis revealed a significantly increased number of myeloid-derived suppressor cells in HSPC-infused mice, which correlated with reduced inflammatory cytokine levels and restored HSPC levels. These findings suggest that HSPCs play an essential immunomodulatory role that may translate into new therapeutic strategies for sepsis.

Outcomes of Human Leukocyte Antigen-Matched Allogeneic Cultivated Limbal Epithelial Transplantation in Aniridia-Associated Keratopathy-A Single-Center Retrospective Analysis.

PURPOSE: To assess the efficacy and safety of human leukocyte antigen-matched allogeneic cultivated limbal epithelial stem cell grafts in the treatment of aniridia-associated keratopathy (AAK). METHODS: Six eyes of 6 patients with severe AAK received an allogeneic stem cell graft between January 2010 and March 2017. Anatomical and functional results were assessed at 6 months, 1 year, 2 years, and the final follow-up visit available. Safety analysis was performed by considering all perioperative and postoperative adverse events and additional surgeries required during the follow-up period. RESULTS: The mean follow-up was 53.6 months (range 24-104 months). In most patients (80%), there was an early improvement of the keratopathy postoperatively, which slowly regressed during longer follow-up. At the final follow-up, 4 of the eyes were graded as failure and 1 eye was graded as partial success. Grading the sixth eye was not possible because of an adverse event. None of the patients maintained a total anatomical success in the long-term. Only 1 patient maintained a modest improvement in best-corrected visual acuity from hand motion to counting fingers. Four serious adverse events were recorded in 2 patients. CONCLUSIONS: Severe AAK remains a challenging condition to manage. Transplantation of allogenic ex vivo cultivated limbal stem cells may provide a temporary improvement in ocular surface stability, but anatomical and functional results are poor in the long-term. The eyes are prone to adverse events, and any surgical treatment should take this into consideration.

An autoimmune stem-like CD8 T cell population drives type 1 diabetes.

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained, and the molecular programmes defining the autoimmune T cell state, are unknown. In type 1 diabetes, ß-cell-specific CD8 T cells destroy insulin-producing ß-cells. Here we followed the fate of ß-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas, where they differentiate further and destroy ß-cells. Whereas transplantation of as few as 20 autoimmune progenitors induced type 1 diabetes, as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived, and stem-like autoimmune progenitors must continuously seed the pancreas to sustain ß-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes.

Stem-cell based, machine learning approach for optimizing natural killer cell-based personalized immunotherapy for high-grade ovarian cancer.

Advanced high-grade serous ovarian cancer continues to be a therapeutic challenge for those affected using the current therapeutic interventions. There is an increasing interest in personalized cancer immunotherapy using activated natural killer (NK) cells. NK cells account for approximately 15% of circulating white blood cells. They are also an important element of the tumor microenvironment (TME) and the body's immune response to cancers. In the present study, DeepNEU-C2Rx, a machine learning platform, was first used to create validated artificially induced pluripotent stem cell simulations. These simulations were then used to generate wild-type artificially induced NK cells (aiNK-WT) and TME simulations. Once validated, the aiNK-WT simulations were exposed to artificially induced high-grade serous ovarian cancer represented by aiOVCAR3. Cytolytic activity of aiNK was evaluated in presence and absence of aiOVCAR3 and data were compared with the literature for validation. The TME simulations suggested 26 factors that could be evaluated based on their ability to enhance aiNK-WT cytolytic activity in the presence of aiOVCAR3. The addition of programmed cell death-1 inhibitor leads to significant reinvigoration of aiNK cytolytic activity. The combination of programmed cell death-1 and glycogen synthase kinase 3 inhibitors showed further improvement. Further addition of ascitic fluid factor inhibitors leads to optimal aiNK activation. Our data showed that NK cell simulations could be used not only to pinpoint novel immunotherapeutic targets to reinvigorate the activity of NK cells against cancers, but also to predict the outcome of targeting tumors with specific genetic expression and mutation profiles.

Cytotoxicity of Reparative Endodontic Cements on Human Periodontal Ligament Stem Cells

ABSTRACT Objective To compare the cytotoxicity of commercial reparative endodontic cements on human periodontal ligament stem cells (hPDLSCs). Material and Methods The culture of hPDLSCs was established. Cell density was set at 2 × 104 cells/well in 96-well plates. Extracts of Biodentine, Bio-C Repair, Cimmo HD, MTA Repair HP and White MTA were prepared. Then, the extracts were diluted (pure, 1:4 and 1:16) and inserted into cell-seeded wells for 24, 48, and 72 h to assess cell viability through MTT assay. hPDLSCs incubated with culture medium alone served as a negative control group. Data were analyzed by Two-Way ANOVA and Tukey's test (α=0.05). Results At 24 h, pure extract of MTA Repair HP and Biodentine 1:16 presented higher cell viability compared to control. Lower cell viability was found for pure extract of Cimmo HD, MTA Repair HP 1:4 and 1:16, and White MTA 1:16. At 48 h, pure extract of Bio-C Repair and MTA Repair HP presented higher cell viability compared to control. At 72 h, only the pure extract of MTA Repair HP led to higher cell proliferation compared to control. Conclusion Biodentine, Bio-C Repair and MTA Repair HP were able to induce hPDLSCs proliferation. Cimmo HD and White MTA were found to be mostly cytotoxic in hPDLSCs.

Genetic Modification of Limbal Stem Cells to Decrease Allogeneic Immune Responses.

Limbal stem cell (LSC) transplantation is the only efficient treatment for patients affected by LSC deficiency (LSCD). Allogeneic LSC transplantation is one of the most successful alternative for patients with bilateral LSCD. Nevertheless, the high variability of the human leukocyte antigens (HLA) remains a relevant obstacle to long-term allogeneic graft survival. This study characterized the immunologic properties of LSCs and proposed a genetic engineering strategy to reduce the immunogenicity of LSCs and of their derivatives. Hence, LSC HLA expression was silenced using lentiviral vectors encoding for short hairpin (sh) RNAs targeting ß2-microglobulin (ß2M) or class II major histocompatibility complex transactivator (CIITA) to silence HLA class I and II respectively. Beside the constitutive expression of HLA class I, LSCs showed the capability to upregulate HLA class II expression under inflammatory conditions. Furthermore, LSCs demonstrated the capability to induce T-cell mediated immune responses. LSCs phenotypical and functional characteristics are not disturbed after genetic modification. However, HLA silenced LSC showed to prevent T cell activation, proliferation and cytotoxicity in comparison to fully HLA-expressing LSCs. Additionally; HLA-silenced LSCs were protected against antibody-mediated cellular-dependent cytotoxicity. Our data is a proof-of-concept of the feasibility to generate low immunogenic human LSCs without affecting their typical features. The use of low immunogenic LSCs may support for long-term survival of LSCs and their derivatives after allogeneic transplantation.

Molecular Mechanisms of Multi-Organ Failure in COVID-19 and Potential of Stem Cell Therapy.

As the number of confirmed cases and deaths occurring from Coronavirus disease 2019 (COVID-19) surges worldwide, health experts are striving hard to fully comprehend the extent of damage caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although COVID-19 primarily manifests itself in the form of severe respiratory distress, it is also known to cause systemic damage to almost all major organs and organ systems within the body. In this review, we discuss the molecular mechanisms leading to multi-organ failure seen in COVID-19 patients. We also examine the potential of stem cell therapy in treating COVID-19 multi-organ failure cases.

Why Are Black People Less Likely to Find a Stem Cell Donor Match?

Building trust in the health care system could increase the number of donors.

Antigen dominance hierarchies shape TCF1+ progenitor CD8 T cell phenotypes in tumors.

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.

Associations Between Inflammation, Cardiovascular Regenerative Capacity, and Cardiovascular Events: A Cohort Study.

Objective: Circulating progenitor cells possess immune modulatory properties and might mitigate inflammation that is characteristic of patients with coronary artery disease. We hypothesized that patients with fewer circulating progenitor cells (CPCs) will have higher inflammatory markers and worse outcomes. Approach and Results: Patients with stable coronary artery disease were enrolled in a prospective study enumerating CPCs as CD (cluster of differentiation)-34-expressing mononuclear cells (CD34+) and inflammation as levels of IL (interleukin)-6 and high-sensitivity CRP (C-reactive protein) levels. Patients were followed for 5 years for the end points of death and myocardial infarction with repeat inflammatory biomarkers measured after a median of 2 years. In the entire cohort of 392 patients, IL-6 and high-sensitivity CRP levels remained unchanged (0.3+/-2.4 pg/mL and 0.1+/-1.0 mg/L; P=0.45) after 2 years. CPC counts (log-transformed) were inversely correlated with the change in IL-6 levels (r, -0.17; P<0.001). Using linear regression, IL-6 and high-sensitivity CRP levels declined by -0.59 (95% CI, -0.90 to -0.20) pg/mL and -0.13 (-0.28 to 0.01) mg/L per 1 log higher CPC counts after adjustment for the demographic and clinical variables, as well as medications. Using Cox models adjusted for these risk factors, a rise in 1 pg/mL of IL-6 was associated with a 11% (95% CI, 9-13) greater risk of death/myocardial infarction. We found that the change in IL6 level partly (by 40%) mediated the higher risk of adverse events among those with low CPC counts. Conclusions: Reduced cardiovascular regenerative capacity is independently associated with progressive inflammation in patients with coronary artery disease that in turn is associated with poor outcomes.

Intrathymic Plasmablasts Are Affected in Patients With Myasthenia Gravis With Active Disease.

BACKGROUND AND OBJECTIVES: To investigate intrathymic B lymphopoiesis in patients with myasthenia gravis (MG) and explore thymus pathology associated with clinical impact. METHODS: Thymic lymphocytes from 15 young patients without MG, 22 adult patients without MG, 14 patients with MG without thymoma, and 11 patients with MG with thymoma were subjected to flow cytometry analysis of T follicular helper (Tfh), naive B, memory B, plasmablasts, CD19+B220high thymic B cells, B-cell activating factor receptor, and C-X-C chemokine receptor 5 (CXCR5). Peripheral blood mononuclear cells of 16 healthy subjects and 21 untreated patients with MG were also analyzed. Immunologic values were compared, and correlations between relevant values and clinical parameters were evaluated. RESULTS: The frequencies of circulating and intrathymic plasmablasts were significantly higher in patients with MG than controls. On the other hand, the frequency of CD19+B220high thymic B cells was not increased in MG thymus. We observed a significant increase in CXCR5 expression on plasmablasts in MG thymus and an increased frequency of intrathymic plasmablasts that was correlated with preoperative disease activity. The frequency of intrathymic Tfh cells was significantly lower in patients who received immunosuppressive (IS) therapy than those without IS therapy. However, there was no significant difference in the frequency of intrathymic plasmablasts irrespective of IS therapy. DISCUSSION: Our findings confirmed a correlation between increased frequency of intrathymic plasmablasts and disease activity before thymectomy. We postulate that activated intrathymic plasmablasts endow pathogenic capacity in MG.

Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer.

T cells are important in tumour immunity but a better understanding is needed of the differentiation of antigen-specific T cells in human cancer1,2. Here we studied CD8 T cells in patients with human papillomavirus (HPV)-positive head and neck cancer and identified several epitopes derived from HPV E2, E5 and E6 proteins that allowed us to analyse virus-specific CD8 T cells using major histocompatibility complex (MHC) class I tetramers. HPV-specific CD8 T cells expressed PD-1 and were detectable in the tumour at levels that ranged from 0.1% to 10% of tumour-infiltrating CD8 T lymphocytes (TILs) for a given epitope. Single-cell RNA-sequencing analyses of tetramer-sorted HPV-specific PD-1+ CD8 TILs revealed three transcriptionally distinct subsets. One subset expressed TCF7 and other genes associated with PD-1+ stem-like CD8 T cells that are critical for maintaining T cell responses in conditions of antigen persistence. The second subset expressed more effector molecules, representing a transitory cell population, and the third subset was characterized by a terminally differentiated gene signature. T cell receptor clonotypes were shared between the three subsets and pseudotime analysis suggested a hypothetical differentiation trajectory from stem-like to transitory to terminally differentiated cells. More notably, HPV-specific PD-1+TCF-1+ stem-like TILs proliferated and differentiated into more effector-like cells after in vitro stimulation with the cognate HPV peptide, whereas the more terminally differentiated cells did not proliferate. The presence of functional HPV-specific PD-1+TCF-1+CD45RO+ stem-like CD8 T cells with proliferative capacity shows that the cellular machinery to respond to PD-1 blockade exists in HPV-positive head and neck cancer, supporting the further investigation of PD-1 targeted therapies in this malignancy. Furthermore, HPV therapeutic vaccination efforts have focused on E6 and E7 proteins; our results suggest that E2 and E5 should also be considered for inclusion as vaccine antigens to elicit tumour-reactive CD8 T cell responses of maximal breadth.

NLRP3 Triggers Attenuate Lipocalin-2 Expression Independent with Inflammasome Activation.

Lipocalin-2 (LCN2), a small secretory glycoprotein, is upregulated by toll-like receptor (TLR) signaling in various cells and tissues. LCN2 inhibits bacterial growth by iron sequestration and regulates the innate immune system. Inflammasome activates the inflammatory caspases leading to pyroptosis and cytokine maturation. This study examined the effects of inflammasome activation on LCN2 secretion in response to TLR signaling. The triggers of NLRP3 inflammasome activation attenuated LCN2 secretion while it induced interleukin-1ß in mouse macrophages. In mice, NLRP3 inflammasome activation inhibited TLR-mediated LCN2 secretion. The inhibition of NLRP3 triggers on LCN2 secretion was caused by the inhibited transcription and translation of LCN2. At the same time, no changes in the other cytokines and IκBζ, a well-known transcriptional factor of Lcn2 transcription, were observed. Overall, NLRP3 triggers are a regulator of LCN2 expression suggesting a new linkage of inflammasome activation and LCN2 secretion in the innate immunity.

Assessment of Immunological Potential of Glial Restricted Progenitor Graft In Vivo-Is Immunosuppression Mandatory?

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease, causing motor neuron and skeletal muscle loss and death. One of the promising therapeutic approaches is stem cell graft application into the brain; however, an immune reaction against it creates serious limitations. This study aimed to research the efficiency of glial restricted progenitors (GRPs) grafted into murine CNS (central nervous system) in healthy models and the SOD1G93A ALS disease model. The cellular grafts were administered in semiallogenic and allogeneic settings. To investigate the models of immune reaction against grafted GRPs, we applied three immunosuppressive/immunomodulatory regimens: preimplantation factor (PiF); Tacrolimus; and CTLA-4, MR1 co-stimulatory blockade. We tracked the cells with bioluminescence imaging (BLI) in vivo to study their survival. The immune response character was evaluated with brain tissue assays and multiplex ELISA in serum and cerebrospinal fluid (CSF). The application of immunosuppressive drugs is disputable when considering cellular transplants into the immune-privileged site/brain. However, our data revealed that semiallogenic GRP graft might survive inside murine CNS without the necessity to apply any immunomodulation or immunosuppression, whereas, in the situation of allogeneic mouse setting, the combination of CTLA-4, MR1 blockade can be considered as the best immunosuppressive option.

Prenatal maternal infection promotes tissue-specific immunity and inflammation in offspring.

The immune system has evolved in the face of microbial exposure. How maternal infection experienced at distinct developmental stages shapes the offspring immune system remains poorly understood. Here, we show that during pregnancy, maternally restricted infection can have permanent and tissue-specific impacts on offspring immunity. Mechanistically, maternal interleukin-6 produced in response to infection can directly impose epigenetic changes on fetal intestinal epithelial stem cells, leading to long-lasting impacts on intestinal immune homeostasis. As a result, offspring of previously infected dams develop enhanced protective immunity to gut infection and increased inflammation in the context of colitis. Thus, maternal infection can be coopted by the fetus to promote long-term, tissue-specific fitness, a phenomenon that may come at the cost of predisposition to inflammatory disorders.

Role of S100A8/A9 for Cytokine Secretion, Revealed in Neutrophils Derived from ER-Hoxb8 Progenitors.

S100A9, a Ca2+-binding protein, is tightly associated to neutrophil pro-inflammatory functions when forming a heterodimer with its S100A8 partner. Upon secretion into the extracellular environment, these proteins behave like damage-associated molecular pattern molecules, which actively participate in the amplification of the inflammation process by recruitment and activation of pro-inflammatory cells. Intracellular functions have also been attributed to the S100A8/A9 complex, notably its ability to regulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. However, the complete functional spectrum of S100A8/A9 at the intracellular level is far from being understood. In this context, we here investigated the possibility that the absence of intracellular S100A8/A9 is involved in cytokine secretion. To overcome the difficulty of genetically modifying neutrophils, we used murine neutrophils derived from wild-type and S100A9-/- Hoxb8 immortalized myeloid progenitors. After confirming that differentiated Hoxb8 neutrophil-like cells are a suitable model to study neutrophil functions, our data show that absence of S100A8/A9 led to a dysregulation of cytokine secretion after lipopolysaccharide (LPS) stimulation. Furthermore, we demonstrate that S100A8/A9-induced cytokine secretion was regulated by the nuclear factor kappa B (NF-κB) pathway. These results were confirmed in human differentiated HL-60 cells, in which S100A9 was inhibited by shRNAs. Finally, our results indicate that the degranulation process could be involved in the regulation of cytokine secretion by S100A8/A9.

Stem cell-like memory T cells: The generation and application.

Stem cell-like memory T cells (Tscm), are a newly defined memory T cell subset with characteristics of long life span, consistent self-renewing, rapid differentiation into effector T cells, and apoptosis resistance. These features indicate that Tscm have great therapeutic or preventive purposes, including being applied in chimeric Ag receptor-engineered T cells, TCR gene-modified T cells, and vaccines. However, the little knowledge about Tscm development restrains their applications. Strength and duration of TCR signaling, cytokines and metabolism in the T cells during activation all influence the Tscm development via regulating transcriptional factors and cell signaling pathways. Here, we summarize the molecular and cellular pathways involving Tscm differentiation, and its clinical application for cancer immunotherapy and prevention.