Cell Activation Markers Research Articles

Blood CD45+/CD3+ lymphocyte-released extracellular vesicles and mortality in hospitalized patients with coronavirus disease 2019.

The global pandemic of coronavirus disease 2019 (COVID-19) represented a major public health concern. Growing evidence shows that plasma of COVID-19 patients contains large numbers of circulating extracellular vesicles (cEVs) that correlate with disease severity and recovery. In this study, we sought to characterize the longitudinal cEV signature in critically ill COVID-19 patients during hospitalization and its relation to mortality risk. cEVs were quantitatively and phenotypically analysed in hospitalized non-surviving COVID-19 patients at baseline (n = 42) and before exitus (n = 40) and in 40 healthy volunteers as a reference group by high sensitivity nano flow cytometry using specific markers for parental cell sources and activation. Levels of cEV subtypes differed between patients with severe COVID-19 and healthy subjects, specifically those from platelets and endothelial, inflammatory and viral infected cells, which associate to high mortality risk. In the longitudinal analysis from baseline to the time point immediately preceding death, no changes were found for platelet, pan-leukocyte, and lung epithelial cell-shed cEVs, while endothelial cell releases of EVs (eEVs) significantly differed. Vascular endothelial growth factor receptor 2-positive eEVs were significantly increased before death compared to admission whereas endoglin and E-selectin-containing eEVs did not change. Conversely, lymphocyte (ℓEV), monocyte, macrophage, pericyte and progenitor cell-derived cEVs displayed significant reductions before exitus. Noteworthy, levels of CD45+/CD3+-ℓEVs were significantly associated to the patient's survival time. An evolving cEV profile able to discriminate prompt risk of death during hospitalization has been defined suggesting a role for circulating and vascular cell-derived EVs in COVID-19 pathogenesis.

Mapping of c-Fos Expression in Rat Brain Sub/Regions Following Chronic Social Isolation: Effective Treatments of Olanzapine, Clozapine or Fluoxetine

The c-Fos as a marker of cell activation is used to identify brain regions involved in stimuli processing. This review summarizes a pattern of c-Fos immunoreactivity and the overlapping brain sub/regions which may provide hints for the identification of neural circuits that underlie depressive- and anxiety-like behaviors of adult male rats following three and six weeks of chronic social isolation (CSIS), relative to controls, as well as the antipsychotic-like effects of olanzapine (Olz), and clozapine (Clz), and the antidepressant-like effect of fluoxetine (Flx) in CSIS relative to CSIS alone. Additionally, drug-treated controls relative to control rats were also characterized. The overlapping rat brain sub/regions with increased expression of c-Fos immunoreactivity following three or six weeks of CSIS were the retrosplenial granular cortex, c subregion, retrosplenial dysgranular cortex, dorsal dentate gyrus, paraventricular nucleus of the thalamus (posterior part, PVP), lateral/basolateral (LA/BL) complex of the amygdala, caudate putamen, and nucleus accumbens shell. Increased activity of the nucleus accumbens core following exposure of CSIS rats either to Olz, Clz, and Flx treatments was found, whereas these treatments in controls activated the LA/BL complex of the amygdala and PVP. We also outline sub/regions that might represent potential neuroanatomical targets for the aforementioned antipsychotics or antidepressant treatments.

CTIM-30. NT-I7, A LONG-ACTING INTERLEUKIN-7, PROMOTES ACTIVATION, CYTOTOXIC FUNCTION, AND SELECTIVE CLONOTYPE EXPANSION OF CD8+ T CELLS IN PATIENTS WITH HIGH GRADE GLIOMAS

Abstract Lymphopenia following chemoradiation for high grade gliomas (HGG) is common and is associated with decreased survival. NT-I7 (efineptakin alfa) is a long-acting analog of the lymphocyte pro-survival cytokine, interleukin-7. Our Phase I dose-escalation study of 19 patients with HGG demonstrated early elevations in CD4+ and CD8+ T cells in the first 4 weeks after administration of NT-I7. In this study, we explored the diversity and potential functionality of the NT-I7-expanded T cell population. A subset of 4 subjects with glioblastoma multiforme (GBM) treated at 720 µg/kg of NT-I7 during adjuvant temozolomide in our study (NCT03687957) underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) of peripheral blood. CITE-seq with TCR clonotype sequencing (10x Genomics 5’) permitted simultaneous tracking of the T cell phenotype at the protein level, individual T cell clones, and gene expression dynamics between 4 timepoints: pre-treatment, and post-treatment weeks 1, 2, and 13. Here, we report the results of differential expression analyses between timepoints and patient-specific differences in T cell clonotype expansions. Both CD4+ and CD8+ T cells in our study patients had increased expression of T cell activation markers LTB, CISH, and CD74. CD8+ T cell subsets demonstrated additional elevations in genes important in cytotoxic function (granzymes (GZMA, GZMB) and perforin (PRF1)), MHC Class II expression (HLA-DRA, HLA-DRB1), and AP-1 transcription factor subunits (JUND, JUN, JUNB, FOS, FOSB). After excluding known viral antigen clonotypes, 2 of 4 patients demonstrated TCR clonotype expansion. Notably, nearly all the expanded TCR clonotypes were found within CD8+ T cells, but not CD4+ T cells. NT-I7 promotes activation, cytotoxic function, and selective clonotype expansion of CD8+ T cells in patients with HGG. This, combined with its role in increased CD4+ T cell counts, suggests NT-I7 may be a powerful tool to restore immune function after treatment-associated lymphopenia in HGG.

Wire injury induced moderate aortic valve stenosis in mice is accompanied by a chronic systemic inflammatory reaction

Abstract Background Patients with aortic valve stenosis (AS) have elevated serum levels of inflammation markers, and blood cytokine levels correlate with ventricular recovery after transcatheter aortic valve replacement. While presence of inflammatory processes in stenotic aortic valves is acknowledged, no systematic characterization of the systemic immune reaction upon AS has been performed, yet. Purpose Hypothesis of this study was that AS induces a systemic inflammatory reaction linked with local processes in the heart. Methods AS was induced by wire injury (WI) or sham surgery was performed in 3-4 months old male C57Bl/6J mice. AS severity, left ventricular (LV) function and hypertrophy indices were assessed with echocardiography (echo). After four weeks, explanted organs were weighted, and flow cytometry identified total leukocyte numbers in blood, heart, and peripheral immune organs (spleen, liver, lymph nodes) and uptake of Cy5-labelled perfluorocarbon nanoemulsions by whole blood leukocytes. Costimulatory molecules (flow cytometry, RT-qPCR) and cytokines (RT-qPCR, Multiplex Immunoassay) were analysed in heart, peripheral immune organs and blood. Results R-values were positive for spleen weight correlation with AS severity, cardiac function and mass suggesting cardiac hypertrophy to preserve heart function. Myocardial myeloid and T cell r-values were positive or negative, respectively, for correlation with spleen and liver weight, hinting at T cell recruitment from peripheral stores. Immune cell numbers in peripheral lymph nodes and spleen showed negative r-values for correlation with heart hypertrophy indices; number of liver myeloid cells correlated negatively with LV wall thickness (monocytes=-0.8571, p=0.0238) suggesting immune cell recruitment to hypertrophic hearts. Cytokine mRNA levels trended mainly towards increase in heart and regional lymph nodes and reduction in spleen and liver after WI. Correlation with echo was more homogeneous after WI, with r-values mainly positive, except of T cell activation markers, for aortic valves, but negative for myocardium, hinting at mechanisms preserving heart function. Correlating unchanged cytokine protein levels in myocardium and plasma with echo, r-values were mostly positive for myocardium, and were more positive than in sham for plasma. Echo and costimulatory molecule correlation showed a more homogeneous pattern in WI, with mostly positive r-values in myocardium and periphery, while most r-values were negative in sham. Together with reduced PFC-uptake by lymphatic cells, this hints at systemic immune cell activation in AS. Conclusion The results suggest that number and activation state of leukocytes in peripheral organs are directly linked to cardiac processes in AS. Considering the pathological value of inflammation, it is crucial to in future studies find out if modulation of the systemic inflammatory reaction relieves severity of AS and prevents development of heart failure.

Astragaloside IV augments anti-PD-1 therapy to suppress tumor growth in lung cancer by remodeling the tumor microenvironment.

Programmed cell death protein-1 (PD-1) inhibitors are increasingly utilized in the treatment of lung cancer (LC). Combination therapy has recently gained popularity in treating LC. This study aimed to assess the efficacy of combining Astragaloside IV (AS-IV) and anti-PD-1 in LC. C57BL/6J mice were subcutaneously injected with Lewis lung carcinoma (LLC) cells. After 3 weeks, the animals were sacrificed, and the tumors were harvested for analysis. Ki-67 immuno-labeling and TUNEL assay were used for evaluating cell proliferation and apoptosis in tumor tissues. In addition, anti-cleaved caspase 3 was used for immunolabelling of apoptotic cells. Immune cell infiltration (macrophages and T cells) and gene expression in tumor tissues were also investigated by using immunofluorescence staining. Compared to treatment with anti-PD-1 or AS-IV, the combination of AS-IV and anti-PD-1 notably reduced tumor volume and weight of LLC-bearing mice. Additionally, the combination treatment strongly induced the apoptosis and suppressed the proliferation in tumor tissues through inactivating PI3K/Akt and ERK signaling pathways, compared to single treatment group. Moreover, the combination treatment elevated levels of the M1 macrophage marker mCD86, reduced levels of the M2 macrophage marker mCD206, as well as upregulated levels of the T cell activation marker mCD69 in tumor tissues. Collectively, the combination treatment effectively inhibited tumor growth in LLC mice through promoting M1 macrophage polarization and T cell activation. These findings showed that combining AS-IV with anti-PD-1 therapy could be a promising therapeutic approach for LC.

Abstract A052: RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction

Abstract RNF43_p.G659fsV*41 mutation (RNF43659mut) is found in approximately 8% of colorectal cancers (CRCs) and is enriched in microsatellite-instability high (MSI-high) tumors. Previously, we reported that RNF43659mut promotes tumor growth independent of WNT signaling and activates PI3K/AKT signaling in CRC, conferring vulnerability to PI3K/MTOR inhibition. In addition, RNF43659mut was associated with decreased interferon signaling suggesting a link with anti-tumor immunity. Thus, in the current work we investigated the role of RNF43659mut in CRC immunomodulation. The immune-cell landscape was evaluated in RNF43659mut compared with RNF43-wild type (wt) MSI-high CRCs in two cohorts; single-cell (sc)-RNA-sequencing data from treatment-naïve primary tumors from 34 patients (cohort-1) and multiplex immunofluorescence / whole exome sequencing data from an independent cohort of 91 patients (cohort-2). Healthy donor blood-derived natural killer (NK) cell responses and cytotoxicity were investigated in co-cultures with RNF43659mut isogenic tumor lines and with patient-derived organoids harboring RNF43659mut or overexpressing RNF43wt. A third patient cohort with MSI-high CRCs treated with immune checkpoint blockade was analyzed to understand the impact of RNF43659mut on immunotherapy efficacy. Interrogation of sc-RNASeq data in cohort-1 revealed a significant enrichment of NK cells in the RNF43659mut patient samples compared to the RNF43wt ones. This finding was also confirmed by multiplex immunofluorescence in cohort-2. Single-cell transcriptomic analyses in cohort-1 showed increased inhibitory markers on NK cells despite their enrichment in RNF43659mut tumors. RNAseq analysis of sorted NK cells co-cultured with RNF43659mut isogenic tumor lines further supported their dysfunctional state. Flow cytometric analyses of NK cell maturation, activation, and inhibitory markers in NK cells co-cultured with RNF43659mut isogenic tumor lines and organoids highlighted decreased levels of NCAM1 (CD56)dim and NCAM1 (CD56)bright subsets, FCGR3A (CD16), and KLRK1 (NKG2D), along with increased levels of KLRC1 (NKG2A), KIR3DL1 (NKB1) and TIGIT inhibitory markers. There was reduced NK cell mediated tumor cell killing in RNF43659mut isogenic tumor lines and organoids. We postulated that aberrant signaling of the PI3K/AKT pathway with RNF43659mut could lead to NK cell dysfunction. Indeed, siRNA-mediated blocking of PI3K/AKT pathway restored the NK-cell dysfunctional phenotype and tumor cell killing in co-cultures with RNF43659mut isogenic tumor lines and organoids. Conversely, overexpression of WT RNF43 in RNF43659mut organoids downregulated PI3K/AKT signaling and increased NK cell cytotoxicity. Lastly, MSI-high CRC tumors harboring RNF43659mut in patients treated with immunotherapy showed less benefit from immune checkpoint blockade. PI3K/AKT activation due to RNF43659mut in MSI-high CRC leads to NK cell dysfunction and reduced NK cell cytotoxicity. This could be responsible for immunotherapy failure, warranting further investigation. Citation Format: Pushpamali De Silva, Samantha Fitzgerald, Jules Cazaubiel, Matan Hofree, Tomotaka Ugai, Juha P Väyrynen, Dane Ford-Roshon, Lishan Fang, Ana Garrido-Castro, Nir Hacohen, Kimmie Ng, Shuji Ogino, Jonathan A Nowak, Marios Giannakis. RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A052.

Transcriptomic analysis of immune-related genes in Pacific white snook (Centropomus viridis) gills infected with the monogenean parasite Rhabdosynochus viridisi

The parasite Rhabdosynochus viridisi (Platyhelminthes: Monogenea) infects the Pacific white snook Centropomus viridis gills and can cause adverse effects in the aquaculture industry. The immune responses of Pacific white snook to monogenean infections are poorly understood. Thus, this study aimed to identify differentially expressed genes (DEGs) in the gills of Pacific white snook juveniles experimentally infected with R. viridisi, emphasizing immune-related genes and pathways activated or suppressed during the infection. RNA sequencing was performed on the gills of uninfected (control) and infected fish. The algorithm Seq2Fun was selected without a reference transcriptome to map the reads to transcripts of fishes available from a database for gene orthologs (EcoOmics) and obtain the counting table. The ExpressAnalyst software was used for differential expression and functional analyses. A total of 20,106 transcripts were found, and 1430 (7 %) were differentially expressed genes (DEGs) between infected and control groups. We identified 860 (60 %) downregulated and 570 (40 %) upregulated genes. Thirteen canonical pathways after the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were overrepresented, and most of the DEGs were downregulated, suggesting the inactivation of these pathways. The functions of most of the DEGs with higher fold change found in this study are poorly understood in fish. Even though the well-known pro-inflammatory cytokines remained unchanged in infected gills of C. viridis, and transforming growth factor β (tgfβ) was downregulated, interleukin-17 ligands il17d and il17a/f1, as well as C-X-C motif chemokine receptor 2 (cxcr2) genes were upregulated, indicating that the infection with R. viridisi promotes Th17-like immunity. Overexpression of plasma B cell activity markers such as immunoglobulin light chain-like genes and the v-set pre-B cell surrogate light chain 3 (vpreb3) was also detected in this study. The possible implications of DEGs related to calcium imbalance, hypoxia adaptation, hemostasis, and immunity are discussed. These results will support future studies to improve the prevention and treatment of monogenean infections in finfish aquaculture.

No signs of mast cell involvement in long-COVID: Acase-control study.

Long-COVID caused by SARS-CoV-2 infection has significant and increasing effects on human health worldwide. Although a unifying molecular or biological explanation is lacking, several pathophysiological mechanisms have been proposed. Involvement of mast cells-evolutionary old "multipurpose" innate immune cells-was reported recently in studies of acute infection and post-acute-COVID-19 syndrome. Mast cell activity has been suggested in long-COVID. In this case-control study, we compared data from 24 individuals with long-COVID (according to the NICE criteria) and 24 age- and sex-matched healthy individuals with a history of SARS-CoV-2 infection without developing sequelae. Serum levels of the proteases beta-tryptase (TPSB2) and carboxypeptidase (CPA3), which are mast cell specific, were measured using immunoassays. The values were compared between the two groups and correlated to measures of physical exertional intolerance. TPSB2 and CPA3 levels were median (range) 26.9 (2.0-1000) and 5.8 (1.5-14.0) ng/mL, respectively, in the long-COVID group. The corresponding values in the control group were 10.9 (2.0-1000) (p = 0.93) and 5.3 (3.5-12.9) ng/mL (p = 0.82). No significant correlations between TPSB2 or CPA3 levels and scores on the ten physical subscales of SF-36, 3.1-3.10 were revealed. We found no significant differences in the levels of mast cell activation markers TPSB2 and CPA3 between the long-COVID and control groups and no correlations with proxy markers of exercise intolerance. Mast cell activation does not appear to be part of long-term pathogenesis of long-COVID, at least in the majority of patients.

Increased levels of serum Adenosine deaminase (ADA) enzyme and increased risk of T cell activation markers in type 2 diabetes

Increased levels of serum Adenosine deaminase (ADA) enzyme and increased risk of T cell activation markers in type 2 diabetes

Abstract P390: The Adipokine Chemerin Does Not Promote Activation or Cytokine Secretion by T Cells Stimulated With a Strong Polyclonal Activator

High fat diet has long been implicated as a risk factor for obesity and other conditions associated with metabolic syndrome, including hypertension. The mechanism by which high fat diet increases risk of hypertension is likely multifactorial, but corresponding low-grade inflammation may play an important role. Low-grade inflammation associated with high fat diet is often accompanied by altered adipokine levels, including an increase in circulating chemerin. However, our understanding of the effects of chemerin and other adipokines on immune cells is limited. Thus, the purpose of these studies was to test the hypothesis that the adipokine chemerin promotes CD4 and CD8 T cell activation and cytokine secretion. To test this hypothesis, primary mouse splenocytes were treated with chemerin or vehicle 30 min prior to activation with a T cell-specific activator (anti-CD3/anti-CD28) for 24 h. T cell activation causes a rapid upregulation of cell surface proteins that are commonly used as experimental markers of activation. We found that chemerin did not impact the induction of the T cell activation markers, CD25, CD69, CD44, CD134, CD137 and CD154, in either CD4 or CD8 T cells. Likewise, we did not see a difference in the downregulation of CD62L, which is rapidly internalized by T cells following activation. Consistent with this, chemerin caused no difference in cytokine secretion by activated T cells. Specifically, the concentrations of IL-2, IL-17A, IFNγ, and TNFα were similar between groups. Overall, this study shows that the adipokine chemerin does not promote acute changes in activation or cytokine expression in primary mouse splenic T cells fully activated with anti-CD3/anti-CD28.

Anti-inflammatory effects of phytocannabinoids and terpenes on inflamed Tregs and Th17 cells in vitro

AimsPhytocannabinoids and terpenes from Cannabis sativa have demonstrated limited anti-inflammatory and analgesic effects in several inflammatory conditions. In the current study, we test the hypothesis that phytocannabinoids exert immunomodulatory effects in vitro by decreasing inflammatory cytokine expression and activation. Key methodsCD3/CD28 and lipopolysaccharide activated peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 6) were treated with phytocannabinoid compounds and terpenes in vitro. Flow cytometry was used to determine regulatory T cell (Treg) and T helper 17 (Th17) cell responses to treatments. Cell pellets were harvested for qRT-PCR gene expression analysis of cytokines, cell activation markers, and inflammation-related receptors. Cell culture supernatants were analysed by ELISA to quantify IL-6, TNF-α and IL-10 secretion. Main findingsIn an initial screen of 20 μM cannabinoids and terpenes which were coded to blind investigators, cannabigerol (GL4a), caryophyllene oxide (GL5a) and gamma-terpinene (GL6a) significantly reduced cytotoxicity and gene expression levels of IL6, IL10, TNF, TRPV1, CNR1, HTR1A, FOXP3, RORC and NFKΒ1. Tetrahydrocannabinol (GL7a) suppression of T cell activation was associated with downregulation of RORC and NFKΒ1 gene expression and reduced IL-6 (p < 0.0001) and IL10 (p < 0.01) secretion. Cannabidiol (GL1b) significantly suppressed activation of Tregs (p < 0.05) and Th17 cells (p < 0.05) in a follow-on in vitro dose-response study. IL-6 (p < 0.01) and IL-10 (p < 0.01) secretion was significantly reduced with 50 μM cannabidiol. SignificanceThe study provides the first evidence that cannabidiol and tetrahydrocannabinol suppress extracellular expression of both anti- and pro-inflammatory cytokines in an in vitro PBMC model of inflammation.

Microglia-specific IL-10 gene delivery inhibits neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease.

Neuroinflammation plays a key role in exacerbating dopaminergic neuron (DAN) loss in Parkinson's disease (PD). However, it remains unresolved how to effectively normalize this immune response given the complex interplay between the innate and adaptive immune responses occurring within a scarcely accessible organ like the brain. In this study, we uncovered a consistent correlation between neuroinflammation, brain parenchymal lymphocytes, and DAN loss among several commonly used mouse models of PD generated by a variety of pathological triggers. We validated a viral therapeutic approach for the microglia-specific expression of interleukin 10 (IL-10) to selectively mitigate the excessive inflammatory response. We found that this approach induced a local nigral IL-10 release that alleviated DAN loss in mice overexpressing the human SNCA gene in the substantia nigra. Single-cell transcriptomics revealed that IL-10 induced the emergence of a molecularly distinct microglial cell state, enriched in markers of cell activation with enhanced expression of prophagocytic pathways. IL-10 promoted microglial phagocytotic and clearance activities in vitro and reduced αSYN aggregate burden in the nigral area in mice overexpressing SNCA. Furthermore, IL-10 stimulated the differentiation of CD4+ T lymphocytes into active T regulatory cells and promoted inhibitory characteristics in CD8+ T cells. In summary, our results show that local and microglia-specific IL-10 transduction elicited strong immunomodulation in the nigral tissue with enhanced suppression of lymphocyte toxicity that was associated with DAN survival. These results offer insights into the therapeutic benefits of IL-10 and showcase a promising gene delivery approach that could minimize undesired side effects.

NLRP3 inflammasome inhibition decreases Schistosomiasis japonica-induced granulomatous inflammation and fibrosis in BALB/c mice.

To research the role of the NLRP3 inflammasome in Schistosoma japonicum-induced granuloma formation and liver fibrosis. In in vivo tests, BALB/c mice were used. shNLRP3 plasmid based on adeno-associated virus serotype 8 (AAV8-shNLRP3) was injected to block NLRP3 inflammasome via tail vein. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected to assess liver injury. H&E staining was used for routine histopathological assessment; Masson's trichrome staining was used to detect fibrous tissues and collagen fibers. Hepatic expression of NLRP3, procaspase-1, bioactive caspase-1, collagen-1, tissue inhibitor of metalloproteinases-1 (TIMP-1), and α-smooth muscle actin (α-SMA) were detected by western blot. Serum levels of IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The inflammatory cell infiltration and hepatic expression of IL-1β around the granuloma were detected by immunohistochemistry staining. Treatment of S. japonicum infected mice with AAV8-shNLRP3 significantly reduced the hepatic levels of bioactive caspase-1 and IL-1β, as well as circulating IL-1β concentrations, while reducing the amounts of myeloperoxidase (MPO) and F4/80 positive cells around the granuloma. Moreover, collagen deposition, TIMP-1, and α-SMA, which are markers of hepatic stellate cell (HSC) activation, were reduced around the liver granuloma. These findings highlight a therapeutic potential of AAV8-shNLRP3 in schistosomiasis cirrhosis.

T cell activation markers CD38 and HLA-DR indicative of non-seroconversion in anti-CD20-treated patients with multiple sclerosis following SARS-CoV-2 mRNA vaccination

BackgroundMessenger RNA (mRNA) vaccines provide robust protection against SARS-CoV-2 in healthy individuals. However, immunity after vaccination of patients with multiple sclerosis (MS) treated with ocrelizumab (OCR), a B cell-depleting anti-CD20...

Immunomodulatory Effects of a Probiotic Mixture: Alleviating Colitis in a Mouse Model through Modulation of Cell Activation Markers and the Gut Microbiota.

Ulcerative colitis (UC) is a persistent inflammatory intestinal disease that consistently affects the colon and rectum. Its exact cause remains unknown. UC causes a considerable challenge in healthcare, prompting research for novel therapeutic strategies. Although probiotics have gained popularity as possible candidates for managing UC, studies are still ongoing to identify the best probiotics or probiotic mixtures for clinical applications. This study aimed to determine the efficacy of a multi-strain probiotic mixture in mitigating intestinal inflammation in a colitis mouse model induced by dextran sulfate sodium. Specifically, a multi-strain probiotic mixture consisting of Tetragenococcus halophilus and Eubacterium rectale was used to study its impact on colitis symptoms. Anti-inflammatory effects were evaluated using ELISA and flow cytometry. The configuration of gut microbial communities was determined using 16S rRNA metagenomic analysis. According to this study, colitis mice treated with the probiotic mixture experienced reduced weight loss and significantly less colonic shortening compared to untreated mice. Additionally, the treated mice exhibited increased levels of forkhead box P3 (Foxp3) and interleukin 10, along with decreased expression of dendritic cell activation markers, such as CD40+, CD80+, and CD83+, in peripheral blood leukocytes and intraepithelial lymphocytes. Furthermore, there was a significant decrease in the frequencies of CD8+N.K1.1+ cells and CD11b+Ly6G+ cells. In terms of the gut microbiota, probiotic-mixture treatment of colitis mice significantly increased the abundance of the phyla Actinobacteria and Verrucomicrobia (p < 0.05). These results provide valuable insights into the therapeutic promise of multi-strain probiotics, shedding light on their potential to alleviate colitis symptoms. This research contributes to the ongoing exploration of effective probiotic interventions for managing inflammatory bowel disease.

Exercise-induced bronchoconstriction in adolescent recreational athletes: Potential screening strategies.

Intense physical exercise in athletes increases the risk to develop exercise-induced bronchocontriction (EIB). We aimed to study EIB prevalence and explore methods for effective EIB screening. Three hundred twenty-seven adolescent athletes (12-18 years) performing at least 12 h of sports a week were included. The evaluation consisted of spirometry, eucapnic voluntary hyperpnoea test (EVH) to evaluate for EIB, FeNO, skin prick testing, blood sampling (serum markers of epithelial damage and mast cell activation), and questionnaires (AQUA©, ACT, ACQ, and exposure and symptom-related questions). Of all athletes, 22% tested positive for EIB (n = 72), 14% reported a previous asthma diagnosis and 40% were atopic. Eighty percent of EIB+ athletes did not use any inhalation therapy. EIB+ athletes were significantly younger, had decreased FEV1/FVC (%), and increased post-EVH-reversibility (%) post-salbutamol compared with EIB- athletes. Furthermore, EIB was significantly associated with previous asthma diagnosis and atopy. The best predictors for a positive EVH test were AQUA© score ≥ 6 (sensitivity of 78%, p = .0171) and wheezing during exercise (specificity of 82%, p = .0002). FeNO negatively and significantly correlated with maximal fall in FEV1 post-EVH test in atopic athletes (r = -.2735, p = .0056). Maximal fall in FEV1 was also associated with prior PM10 exposure (p = .036). Serum markers of epithelial damage were significantly associated with training type, training intensity, EIB severity, and prior air pollution exposure. Our findings support the effectiveness of a systematic respiratory screening approach, including baseline questionnaires, lung function tests, and FeNO measurement, to improve EIB detection in adolescent athletes in whom respiratory response to EVH testing is associated with prior exposure to air pollution.

CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

Baseline Blood CD8+ T Cell Activation Potency Discriminates Responders from Non-Responders to Immune Checkpoint Inhibition Combined with Stereotactic Radiotherapy in Non-Small-Cell Lung Cancer.

Tumor-infiltrating immune cells have been correlated with prognosis for patients treated with immune checkpoint inhibitor (ICI) treatment of various cancers. However, no robust biomarker has been described to predict treatment response yet. We hypothesized that the activation potency of circulating T cells may predict response to ICI treatment. An exploratory analysis was conducted to investigate the association between the response to immune checkpoint inhibition (ICI) combined with stereotactic radiotherapy (SBRT) and the potency of circulating T cells to be activated. Blood-derived lymphocytes from 14 patients were stimulated ex vivo with, among others, Staphylococcal enterotoxin B (SEB) and compared to healthy controls (HCs). Patients were grouped into responders (>median progression free survival (PFS)) and non-responders (<median PFS). The expression of the T cell activation marker CD69 and intracellular cytokines (IL-2, IFNγ, TNFα) in both CD4+ and CD8+ T cells in response to stimulation was measured using flow cytometry. In addition, serum levels of BAFF, IFNγ, and IL-2 receptor (sIL-2R) were measured by Luminex. At baseline, a higher percentage of activated CD8+ T cells (15.8% vs. 3.5% (p = <0.01)) and IL-2+CD69+CD8+ T cells (8.8% vs. 2.9% (p = 0.02)) was observed in responders compared to non-responders upon ex vivo stimulation with SEB. The concurrently measured serum cytokine levels were not different between responders and non-responders. Baseline blood CD8+ T cell activation potency, measured by intracellular cytokine production after ex vivo stimulation, is a potential biomarker to discriminate responders from non-responders to SBRT combined with ICI.

Ritlecitinib, a JAK3/TEC family kinase inhibitor, stabilizes active lesions and repigments stable lesions in vitiligo

The efficacy of ritlecitinib, an oral JAK3/TEC family kinase inhibitor, on active and stable lesions was evaluated in patients with active non-segmental vitiligo in a phase 2b trial (NCT03715829). Patients were randomized to placebo or daily ritlecitinib 50 mg (with or without 4-week 100-mg or 200-mg loading dose), 30 mg, or 10 mg for 24 weeks. Active lesions showed greater baseline expression of inflammatory/immune markers IFNG and CCL5, levels of CD103, and T-cell infiltrates than stable lesions. Patients with more active than stable vitiligo lesions showed higher baseline serum levels of CXCL9 and PD-L1, while patients with more stable than active lesions showed higher baseline serum levels of HO-1. At Week 24, ritlecitinib 50 mg significantly stabilized mean percent change from baseline in depigmentation extent in both active lesions and stable lesions vs. placebo-response, with stable lesions showing greater repigmentation. After 24 weeks of treatment, ritlecitinib 50 mg increased expression of melanocyte markers in stable lesions, while Th1/Th2-related and co-stimulatory molecules decreased significantly in both stable and active lesions. Serum from patients with more active than stable lesions showed decreased levels of ICOS and NK cell activation markers. These data, confirmed at transcription/protein levels, indicate that stable lesion repigmentation occurs early with ritlecitinib, while active lesions require stabilization of inflammation first. ClinicalTrials.gov: NCT03715829.

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.