Arming AAV9 with a Single-Chain Fragment Variable Antibody Against PD-1 for Systemic Glioblastoma Therapy.

Glioblastoma (GBM) is a highly aggressive brain cancer with a low survival rate, prompting the exploration of novel therapeutic strategies. Immune checkpoint inhibitors have shown promise in cancer treatment but are associated with immune-related toxicities and brain penetration. Here, we present a targeted approach using an adeno-associated virus serotype 9 (AAV9) to systemically deliver a single-chain fragment variable antibody against PD-1 (scFv-PD-1) into the tumor microenvironment (TME). Single-cell RNA sequencing analysis revealed robust PD-1 expression in GBM TME, predominantly on T cells. AAV9-scFv-PD-1 expressed and secreted scFv-PD-1, which effectively binds to PD-1. Systemic administration of AAV9-scFv-PD-1 in an immunocompetent GBM mouse model resulted in a robust cytolytic T-cell activation at the tumor site, marked by accumulation of IFN-γ and Granzyme B, leading to a significant reduction in tumor growth. Importantly, AAV9-scFv-PD-1 treatment conferred a survival benefit, highlighting its therapeutic potential. This study demonstrates the feasibility of systemically delivered AAV9-mediated local expression of scFv-PD-1 for targeted immunotherapy in GBM and warrants further investigation for clinical translation.

Targeting the MHC-I endosomal-lysosomal trafficking pathway in cancer: From mechanism to immunotherapy.

Immune checkpoint blockade (ICB) therapy has achieved broad applicability and durable clinical responses across cancer types. However, the overall response rate remains suboptimal because some patients do not respond or develop drug resistance. The low infiltration of CD8+ cytotoxic T cells (CTLs) in the tumor microenvironment due to insufficient antigen presentation is closely related to the innate resistance to ICB. The duration and spatial distribution of major histocompatibility complex class I (MHC-I) expression on the cell surface is critical for the efficient presentation of endogenous tumor antigens and subsequent recognition and clearance by CTLs. Tumor cells reduce the surface expression of MHC-I via multiple mechanisms to impair antigen presentation pathways and evade immunity and/or develop resistance to ICB therapy. As an increasing number of studies have focused on membrane MHC-I trafficking and degradation in tumor cells, which may impact the effectiveness of tumor immunotherapy. It is necessary to summarize the mechanism regulating membrane MHC-I translocation into the cytoplasm and degradation via the lysosome. We reviewed recent advances in the understanding of endosomal-lysosomal MHC-I transport and highlighted the means exploited by tumor cells to evade detection and clearance by CTLs. We also summarized new therapeutic strategies targeting these pathways to enhance classical ICB treatment and provide new avenues for optimizing cancer immunotherapy.

High Cellular Internalization of Virus-Like Mesoporous Silica Nanoparticles Enhances Adaptive Antigen-Specific Immune Responses against Cancer.

Effective activation of an antigen-specific immune response hinges upon the intracellular delivery of cancer antigens to antigen-presenting cells (APCs), marking the initial stride in cancer vaccine development. Leveraging biomimetic topological morphology, we employed virus-like mesoporous silica nanoparticles (VMSNs) coloaded with antigens and toll-like receptor 9 (TLR9) agonists to craft a potent cancer vaccine. Our VMSNs could be efficiently internalized by APCs to a greater extent than their nonviral structured counterparts, thereby promoting the activation of APCs by upregulating the TLR9 pathway and cross-presenting ovalbumin (OVA) epitopes. In in vivo animal study, VMSN-based nanovaccines triggered substantial CD4+ and CD8+ lymphocyte populations in both lymph nodes and spleen while inducing the effector memory of adaptive T cells. Consequently, VMSN-based nanovaccines suppressed tumor progression and increased the survival rate of B16-OVA-bearing mice in both prophylactic and therapeutic studies. The combination of immune checkpoint blockade (ICB) with the VMSN-based nanovaccine has synergistic effects in significantly preventing tumor progression under therapeutic conditions. These findings highlight the potential of viral structure-mimicking mesoporous silica nanoparticles as promising candidates for antigen-delivering nanocarriers in vaccine development.

Vaccination of cattle with a virus vector vaccine against a major membrane protein of Mycobacterium avium subsp. paratuberculosis elicits CD8 cytotoxic T cells that kill intracellular bacteria.

Analysis of the recall response ex vivo in cattle vaccinated with a Mycobacterium avium subsp. paratuberculosis (Map) rel deletion mutant revealed the immune response was directed toward a 35 kD major membrane protein (MMP) of Map. Antigen presenting cells (APC) primed with MMP elicited expansion of CD8 cytotoxic memory T cells (CTL) with ability to kill intracellular bacteria. Development of CTL was MHC-restricted. The gene MAP2121c, encoding MMP, was modified for expression of MMP (tPA-MMP-2mut) in a mammalian cell line to explore the potential of developing MMP as a vaccine. Ex vivo stimulation of PBMC, from Map free cattle, with APC primed with tPA-MMP-2mut expressed p35 elicited a primary CD8 CTL response comparable to the recall response elicited with PBMC from cattle vaccinated with either the Maprel deletion mutant or MMP. In the present study, the modified gene for MMP, now referred to as p35NN, was placed into a bovine herpes virus-4 (BoHV4) vector to determine the potential use of BoHV-4AΔTK-p35NN as a peptide-based vaccine. Subcutaneous vaccination of healthy cattle with BoHV-4AΔTK-p35NN elicited a CTL recall response, as detected ex vivo. The results show use of a virus vector is an effective way for delivery of MMP as a vaccine. The immunogenic activity of MMP was not lost when modified for expression in mammalian cells. The next step is to conduct a field trial to determine if presence of an immune response to MMP prevents Map from establishing an infection.

EBV Reactivation and Disease in Allogeneic Hematopoietic Stem Cell Transplant (HSCT) Recipients and Its Impact on HSCT Outcomes.

The acquisition or reactivation of Epstein-Barr virus (EBV) after allogeneic Hematopoietic Stem Cell Transplant (HSCT) can be associated with complications including the development of post-transplant lymphoproliferative disorder (PTLD), which is associated with significant morbidity and mortality. A number of risk factors for PTLD have been defined, including T-cell depletion, and approaches to monitoring EBV, especially in high-risk patients, with the use of preemptive therapy upon viral activation have been described. Newer therapies for the preemption or treatment of PTLD, such as EBV-specific cytotoxic T-cells, hold promise. Further studies to help define risks, diagnosis, and treatment of EBV-related complications are needed in this at-risk population.

Diversity of Memory CD8+ T Cells in Tumor-Draining Lymph Nodes from Patients with Bladder Cancer.

The role of memory T cells in orchestrating memory responses to previously known tumor antigens is well documented. The aim of this study was to assess the frequency of different memory T cell subsets in tumor-draining lymph nodes of patients with bladder cancer (BC) and their prognostic significance. Mononuclear cells were isolated from 50 tumor-draining lymph nodes of untreated patients with BC and stained with antibodies against the markers CD8, CD95, CD45RO and CCR7. Data were collected using the FACSCalibur flow cytometer and analyzed using FlowJo software. Among the CD8+ cytotoxic lymphocytes, the frequency of different subsets was determined including total memory cells (CD8+CD45RO+CD95+), T central memory (TCM: CD8+CCR7+CD45RO+CD95+), T effector memory (TEM: CD8+CCR7-CD45RO+CD95+), T stem cell memory (TSCM: CD8+CCR7+CD45RO-CD95+) and naïve T cells (CD8+CCR7+CD45RO-CD95-). The analysis revealed that on average 49.32±20.15 (between 1.62% and 87.20%) percent of CD8+ lymphocytes in draining lymph nodes of BC had a memory phenotype. TCM cells showed the highest frequency (34.71±17.04), while TSCM cells (7.51±8.53) demonstrated the lowest. The total frequency of memory cells tended to be higher in patients with tumor invasion to muscle layer (P=0.052) and stage III (P=0.042) than in patients without invasion and stage I. The TCM subset was more frequent in patients with necrotic tumors than in patients without necrosis (P=0.048). TSCM significantly increased in patients with N2 compared to N0 (P=0.042). Conversely, the ratio of TSCM cells to total memory cells was higher in lower tumor stages (P=0.059), tumors without muscle invasion (P=0.026) and low T grouping (P=0.043). Overall the data indicated an increase in the frequency of memory T cells and their TSCM and TCM cells with tumor progression. In contrast, the ratio of TSCM to total memory cells was higher in less advanced tumors. These results suggest that the immune system is frequently exposed to tumor antigens and strives to create a memory T cell reservoir, but this is suppressed by inhibitory factors provided by the tumor. These findings emphasize the importance of understanding the dynamic interplay between memory T cell subsets and BC progression.

Engineered HCMV-infected APCs enable the identification of new immunodominant HLA-restricted epitopes of anti-HCMV T-cell immunity.

Complications due to HCMV infection or reactivation remain a challenging clinical problem in immunocompromised patients, mainly due to insufficient or absent T-cell functionality. Knowledge of viral targets is crucial to improve monitoring of high-risk patients and optimise antiviral T-cell therapy. To expand the epitope spectrum, genetically-engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble (s)HLA-A*11:01 and infected with an HCMV mutant lacking immune evasion molecules (US2-6 + 11). More than 700 HLA-A*11:01-restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open-reading-frames (ORFs) were identified by mass spectrometry and screened for HLA-A*11:01-binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy HLA-A*11:01+/HCMV+ donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti-apoptotic protein UL36 and ORFL101C (A11SAL), were identified. Their HLA-A*11:01 complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T-cell responses against A11SAL, with T-cell responses comparable to the immunodominant HLA-A*02:01-restricted HCMVpp65NLV epitope. A11SAL-specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV-infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically-engineered DCs to identify naturally presented immunodominant HCMV-derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options.

Adaptive Cellular Responses following SARS-CoV-2 Vaccination in Primary Antibody Deficiency Patients.

Since the start of the COVID-19 pandemic, in a short span of 3 years, vaccination against SARS-CoV-2 has resulted in the end of the pandemic. Patients with inborn errors of immunity (IEI) are at an increased risk for SARS-CoV-2 infection; however, serious illnesses and mortality, especially in primary antibody deficiencies (PADs), have been lower than expected and lower than other high-risk groups. This suggests that PAD patients may mount a reasonable effective response to the SARS-CoV-2 vaccine. Several studies have been published regarding antibody responses, with contradictory reports. The current study is, perhaps, the most comprehensive study of phenotypically defined various lymphocyte populations in PAD patients following the SARS-CoV-2 vaccine. In this study, we examined, following two vaccinations and, in a few cases, prior to and following the 1st and 2nd vaccinations, subsets of CD4 and CD8 T cells (Naïve, TCM, TEM, TEMRA), T follicular helper cells (TFH1, TFH2, TFH17, TFH1/17), B cells (naïve, transitional, marginal zone, germinal center, IgM memory, switched memory, plasmablasts, CD21low), regulatory lymphocytes (CD4Treg, CD8Treg, TFR, Breg), and SARS-CoV-2-specific activation of CD4 T cells and CD8 T cells (CD69, CD137), SARS-CoV-2 tetramer-positive CD8 T cells, and CD8 CTL. Our data show significant alterations in various B cell subsets including Breg, whereas only a few subsets of various T cells revealed alterations. These data suggest that large proportions of PAD patients may mount significant responses to the vaccine.

Sex differences in donor T cell targeting of host splenocyte subpopulations in acute and chronic murine graft-vs.-host disease: implications for lupus-like autoimmunity.

This study sought to compare in vivo sex differences in either a Th1-dominant CTL response or a Tfh-mediated lupus-like antibody response using the parent-into F1 murine model of acute or chronic GVHD respectively. In acute GVHD we observed no significant sex differences in the hierarchy of donor CD8 CTL elimination of splenocyte subsets. B cells were the most sensitive to elimination in both sexes; however, the male response was significantly stronger. Sex differences in chronic GVHD were more widespread; females exhibited significantly greater numbers of total splenocytes and host CD4 Tfh cells, B cells and CD8 T cells consistent with reports of greater female autoantibody production in this model. The more potent male CTL response in acute GVHD conflicts with reports of greater female CTL responses following infections or vaccines and may reflect the absence of exogenous innate immune stimuli in this model.

Evaluation of ruxolitinib cream 1.5% as an at-home therapy for repigmentation in non-segmental vitiligo.

INTRODUCTION: Vitiligo is a chronic, autoimmune condition characterized by skin depigmentation caused by inflammatory-mediated melanocyte degradation. Treatment of vitiligo is challenging due to the chronic nature of the condition. Ruxolitinib cream 1.5% was recently approved by the Food and Drug Administration (FDA) as a Janus kinase 1 and 2 inhibitor for use in nonsegmental vitiligo for those 12 years and older. AREAS COVERED: The purpose of this review is to describe the role of ruxolitinib in treating nonsegmental vitiligo.We searched PubMed using search terms nonsegmental vitiligo, jak inhibitor, and ruxolitinib. Clinicaltrials.gov was used to identify clinical trial data including efficacy, pharmacodynamics, pharmacokinetics, safety, and tolerability. EXPERT OPINION: In both phase II and phase III (TRuE-V1 and TRuE-V2) trials, ruxolitinib cream 1.5% improved repigmentation with minimal adverse effects. Topical ruxolitinib is a much needed new vitiligo treatment option.  Real life efficacy may not match that seen in clinical trials if the hurdle of poor adherence to topical treatment is not surmounted.

The CD8+ T cell content of transbronchial biopsies from patients with a first episode of clinically stable grade A1 cellular rejection is associated with future chronic lung allograft dysfunction.

BACKGROUND: T cells drive acute cellular rejection (ACR) and its progression to chronic lung allograft dysfunction (CLAD) following lung transplantation. International Society for Heart and Lung Transplantation grade A1 ACR without associated allograft dysfunction is often untreated, yet some patients develop progressive graft dysfunction. T-cell composition of A1 ACR lesions may have prognostic value; therefore, protein-level and epigenetic techniques were applied to transbronchial biopsy tissue to determine whether differential T-cell infiltration in recipients experiencing a first episode of stable grade A1 ACR (StA1R) is associated with early CLAD. METHODS: Sixty-two patients experiencing a first episode of StA1R were divided into those experiencing CLAD within 2 years (n = 13) and those remaining CLAD-free for 5 or more years (n = 49). Imaging mass cytometry (IMC) was used to profile the spectrum and distribution of intragraft T cell phenotypes on a subcohort (n = 16; 8 early-CLAD and 8 no early-CLAD). Immunofluorescence was used to quantify CD4+, CD8+, and FOXP3+ cells. Separately, CD3+ cells were fluorescently labeled, micro-dissected, and the degree of Treg-specific demethylated region methylation was determined. RESULTS: PhenoGraph unsupervised clustering on IMC revealed 50 unique immune cell subpopulations. Methylation and immunofluorescence analyses demonstrated no significant differences in Tregs between early-CLAD and no early-CLAD groups. Immunofluorescence revealed that patients who developed CLAD within 2 years of lung transplantation showed greater CD8+ T cell infiltration compared to those who remained CLAD-free for 5 or more years. CONCLUSIONS: In asymptomatic patients with a first episode of A1 rejection, greater CD8+ T cell content may be indicative of worse long-term outlook.

Factor of time in dendritic cell (DC) maturation: short-term activation of DCs significantly improves type 1 cytokine production and T cell responses.

Dendritic cells (DCs) have been intensively studied in correlation to tumor immunology and for the development DC-based cancer vaccines. Here, we present the significance of the temporal aspect of DC maturation for the most essential subsequent timepoint, namely at interaction with responding T cells or after CD40-Ligand restimulation. Mostly, DC maturation is still being achieved by activation processes which lasts 24 h to 48 h. We hypothesized this amount of time is excessive from a biological standpoint and could be the underlying cause for functional exhaustion. Indeed, shorter maturation periods resulted in extensive capacity of monocyte-derived DCs to produce inflammatory cytokines after re-stimulation with CD40-Ligand. This effect was most evident for the primary type 1 polarizing cytokine, IL-12p70. This capacity reached peak at 6 h and dropped sharply with longer exposure to initial maturation stimuli (up to 48 h). The 6 h maturation protocol reflected superiority in subsequent functionality tests. Namely, DCs displayed twice the allostimulatory capacity of 24 h- and 48 h-matured DCs. Similarly, type 1 T cell response measured by IFN-γ production was 3-fold higher when CD4+ T cells had been stimulated with shortly matured DC and over 8-fold greater in case of CD8+ T cells, compared to longer matured DCs. The extent of melanoma-specific CD8+ cytotoxic T cell induction was also greater in case of 6 h DC maturation. The major limitation of the study is that it lacks in vivo evidence, which we aim to examine in the future. Our findings show an unexpectedly significant impact of temporal exposure to activation signals for subsequent DC functionality, which we believe can be readily integrated into existing knowledge on in vitro/ex vivo DC manipulation for various uses. We also believe this has important implications for DC vaccine design for future clinical trials.

In situ analysis of CCR8+ regulatory T cells in lung cancer: suppression of GzmB+ CD8+ T cells and prognostic marker implications.

BACKGROUND: CCR8-expressing regulatory T cells (Tregs) are selectively localized within tumors and have gained attention as potent suppressors of anti-tumor immunity. This study focused on CCR8+ Tregs and their interaction with CD8+ T cells in the tumor microenvironment of human lung cancer. We evaluated their spatial distribution impact on CD8+ T cell effector function, specifically granzyme B (GzmB) expression, and clinical outcomes. METHODS: A total of 81 patients with lung squamous cell carcinoma (LSCC) who underwent radical surgical resection without preoperative treatment were enrolled. Histological analyses were performed, utilizing an automated image analysis system for double-stained immunohistochemistry assays of CCR8/Foxp3 and GzmB/CD8. We investigated the association of CCR8+ Tregs and GzmB+ CD8+ T cells in tumor tissues and further evaluated the prognostic impact of their distribution profiles. RESULTS: Histological evaluation using the region of interest (ROI) protocol showed that GzmB expression levels in CD8+ T cells were decreased in areas with high infiltration of CCR8+ Tregs, suggesting a suppressive effect of CCR8+ Tregs on T cell cytotoxicity in the local tumor microenvironment. Analysis of the association with clinical outcomes showed that patients with more CCR8+ Tregs and lower GzmB expression, represented by a low GzmB/CCR8 ratio, had worse progression-free survival. CONCLUSIONS: Our data suggest that local CCR8+ Treg accumulation is associated with reduced CD8+ T cell cytotoxic activity and poor prognosis in LSCC patients, highlighting the biological role and clinical significance of CCR8+ Tregs in the tumor microenvironment. The GzmB/CCR8 ratio may be a useful prognostic factor for future clinical applications in LSCC.

Haplo-insufficiency of Profilin1 in vascular endothelial cells is beneficial but not sufficient to confer protection against experimentally induced atherosclerosis.

Actin cytoskeleton plays an important role in various aspects of atherosclerosis, a key driver of ischemic heart disease. Actin-binding protein Profilin1 (Pfn1) is overexpressed in atherosclerotic plaques in human disease, and Pfn1, when partially depleted globally in all cell types, confers atheroprotection in vivo. This study investigates the impact of endothelial cell (EC)-specific partial loss of Pfn1 expression in atherosclerosis development. We utilized mice engineered for conditional heterozygous knockout of the Pfn1 gene in ECs, with atherosclerosis induced by depletion of hepatic LDL receptor by gene delivery of PCSK9 combined with high-cholesterol diet. Our studies show that partial depletion of EC Pfn1 has certain beneficial effects marked by dampening of select pro-atherogenic cytokines (CXCL10 and IL7) with concomitant reduction in cytotoxic T cell abundance but is not sufficient to reduce hyperlipidemia and confer atheroprotection in vivo. In light of these findings, we conclude that atheroprotective phenotype conferred by global Pfn1 haplo-insufficiency requires contributions of additional cell types that are relevant for atherosclerosis progression.

Urolithin A Hijacks ERK1/2-ULK1 Cascade to Improve CD8+ T Cell Fitness for Antitumor Immunity.

According to the latest evidence, the microbial metabolite Urolithin A (UA), known for its role in promoting cellular health, modulates CD8+ T cell-mediated antitumor activity. However, the direct target protein of UA and its underlying mechanism remains unclear. Here, this research identifies ERK1/2 as the specific target crucial for UA-mediated CD8+ T cell activation. Even at low doses, UA markedly enhances the persistence and effector functions of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells both in vitro and in vivo. Mechanistically, UA interacts directly with ERK1/2 kinases, enhancing their activation and subsequently facilitating T cell activation by engaging ULK1. The UA-ERK1/2-ULK1 axis promotes autophagic flux in CD8+ CTLs, enhancing cellular metabolism and maintaining reactive oxygen species (ROS) levels, as evidenced by increased oxygen consumption and extracellular acidification rates. UA-treated CD8+ CTLs also display elevated ATP levels and enhanced spare respiratory capacity. Overall, UA activates ERK1/2, inducing autophagy and metabolic adaptation, showcasing its potential in tumor immunotherapy and interventions for diseases involving ERKs.

Cytotoxic response of tumor-infiltrating lymphocytes of head and neck cancer slice cultures under mitochondrial dysfunction.

Background: Head and neck squamous cell carcinomas (HNSCC) are highly heterogeneous tumors. In the harsh tumor microenvironment (TME), metabolic reprogramming and mitochondrial dysfunction may lead to immunosuppressive phenotypes. Aerobic glycolysis is needed for the activation of cytotoxic T-cells and the absence of glucose may hamper the full effector functions of cytotoxic T-cells. To test the effect of mitochondrial dysfunction on cytotoxic T cell function, slice cultures (SC) of HNSCC cancer were cultivated under different metabolic conditions. Methods: Tumor samples from 21 patients with HNSCC were collected, from which, SC were established and cultivated under six different conditions. These conditions included high glucose, T cell stimulation, and temporarily induced mitochondrial dysfunction (MitoDys) using FCCP and oligomycin A with or without additional T cell stimulation, high glucose and finally, a control medium. Over three days of cultivation, sequential T cell stimulation and MitoDys treatments were performed. Supernatant was collected, and SC were fixed and embedded. Granzyme B was measured in the supernatant and in the SC via immunohistochemistry (IHC). Staining of PD1, CD8/Ki67, and cleaved-caspase-3 (CC3) were performed in SC. Results: Hematoxylin eosin stains showed that overall SC quality remained stable over 3 days of cultivation. T cell stimulation, both alone and combined with MitoDys, led to significantly increased granzyme levels in SC and in supernatant. Apoptosis following T cell stimulation was observed in tumor and stroma. Mitochondrial dysfunction alone increased apoptosis in tumor cell aggregates. High glucose concentration alone had no impact on T cell activity and apoptosis. Apoptosis rates were significantly lower under conditions with high glucose and MitoDys (p=0.03). Conclusion: Stimulation of tumor-infiltrating lymphocytes in SC was feasible, which led to increased apoptosis in tumor cells. Induced mitochondrial dysfunction did not play a significant role in the activation and function of TILs in SC of HNSCC. Moreover, high glucose concentration did not promote cytotoxic T cell activity in HNSCC SC.

EBV infected cells in the multiple sclerosis brain express PD-L1: How the virus and its niche may escape immune surveillance.

The presence of EBV infected B cells in postmortem multiple sclerosis (MS) brain tissue suggests immune evasion strategies. Using immunohistochemical techniques we analysed the expression of the immune checkpoint molecule PD-L1 and its receptor PD-1 in MS brains containing B cell-enriched perivascular infiltrates and meningeal follicles, a major EBV reservoir. PD-1 and PD-L1 immunoreactivities were restricted to CNS-infiltrating immune cells. PD-L1 was expressed on B cells, including EBV infected B cells, while PD-1 was expressed on many CD8+ T cells, including EBV-specific CD8+ T-cells, and fewer CD4+ T cells. PD-L1+ cells and EBV infected cells were in close contact with PD-1+ T cells. PD-L1 expressed by EBV infected B cells could favour local immune evasion leading to EBV persistence and immunopathology in the MS brain.

T-cell immune profile in blood of systemic mastocytosis: Association with disease features.

BACKGROUND: Systemic mastocytosis (SM) is a heterogeneous disease characterized by an expansion of KIT-mutated mast cells (MC). KIT-mutated MC display activated features and release MC mediators that might act on the tumour microenvironment and other immune cells. Here, we investigated the distribution of lymphocyte subsets in blood of patients with distinct subtypes of SM and determined its association with other disease features. METHODS: We studied the distribution of TCD4+ and TCD4- cytotoxic cells and their subsets, as well as total NK- and B cells, in blood of 115 SM patients-38 bone marrow mastocytosis (BMM), 67 indolent SM (ISM), 10 aggressive SM (ASM)- and 83 age-matched healthy donors (HD), using spectral flow cytometry and the EuroFlow Immunomonitoring panel, and correlated it with multilineage KITD816V, the alpha-tryptasemia genotype (HαT) and the clinical manifestations of the disease. RESULTS: SM patients showed decreased counts (vs. HD) of TCD4- cytotoxic cells, NK cells and several functional subsets of TCD4+ cells (total Th1, Th2-effector memory, Th22-terminal effector and Th1-like Tregs), together with increased T-follicular-helper and Th1/Th17-like Treg counts, associated with different immune profiles per diagnostic subtype of SM, in multilineal versus MC-restricted KITD816V and in cases with a HαT+ versus HαT- genotype. Unique immune profiles were found among BMM and ISM patients with MC-restricted KITD816V who displayed HαT, anaphylaxis, hymenoptera venom allergy, bone disease, pruritus, flushing and GI symptoms. CONCLUSION: Our results reveal altered T- and NK-cell immune profiles in blood of SM, which vary per disease subtype, the pattern of involvement of haematopoiesis by KITD816V, the HαT genotype and specific clinical manifestations of the disease.

Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cells.

Actin cytoskeleton remodeling sustains the ability of cytotoxic T cells to search for target cells and eliminate them. We here investigated the relationship between energetic status, actin remodeling, and functional fitness in human CD8+ effector T cells. Cell spreading during migration or immunological synapse assembly mirrored cytotoxic activity. Morphological and functional fitness were boosted by interleukin-2 (IL-2), which also stimulated the transcription of glycolytic enzymes, actin isoforms, and actin-related protein (ARP)2/3 complex subunits. This molecular program scaled with F-actin content and cell spreading. Inhibiting glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility, and adhesion, while mitochondrial oxidative phosphorylation blockade impacted cell elongation during confined migration. The severe morphological and functional defects of ARPC1B-deficient T cells were only partially corrected by IL-2, emphasizing ARP2/3-mediated actin polymerization as a crucial energy state integrator. The study therefore underscores the tight coordination between metabolic and actin remodeling programs to sustain the cytotoxic activity of CD8+ T cells.

Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection.

Background: Helicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection. Methods: We analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines. Results: In biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells. Conclusion: H. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy.