Frequency Of NKT Cells Research Articles

Analysis of the Blood Levels of NK and NKT Cells in Patients with Severe SARS-CoV-2 Infection.

Clinical features of SARS-CoV-2 infection vary, ranging from asymptomatic cases to pneumonia, and other serious complications. Some populations have been observed to be at higher risk for severe disease and death compared to other ethnical groups. To evaluate two parameters of the innate immune system, that play a significant role in viral immunity. In samples of peripheral blood from sixteen patients with severe COVID-19, ten with asymptomatic to mild illness, and fifteen healthy subjects, the percentage of NK and NKT cells, the expression of different NK cell receptors and the blood levels of pro-inflammatory cytokines were tested. We observed that patients with severe COVID-19 showed significantly lower frequencies of both CD56dim and CD56bright NK cells compared to patients with mild illness or healthy controls. Furthermore, patients with severe manifestation of COVID-19 exhibited an aberrant expression of the natural cytotoxicity receptors NKp30, NKp44 and NKp46. Similarly, NK cells from these patients showed statistically significant differences in the expression of various killer immunoglobulin-like receptors (KIRs) in the two main cell subsets (CD56bright, CD56dim) compared to controls or patients with mild disease. Moreover, patients with severe illness displayed decreased frequency of NKT cells (defined as CD3+CD56+) and elevated blood levels of the cytokines IL-6 and IL-8. This study suggests that the abnormal features of NK and NKT cells observed in patients with severe SARS-CoV-2 infection may play an important role in the outcome of this infectious disease in various population groups.

DOP19 High dimensional profiling of IL-23-responsive cells revealed molecular signatures that predict response to anti-IL-23 therapy in patients with Crohn’s Disease

Abstract Background The interleukin (IL)-23 signaling plays a crucial role in the pathogenesis of Crohn’s disease (CD). IL-23 activates T-helper (Th) 17 cells, IL-17-secreting CD8 T (Tc)17 cells, gd T cells, nature killer (NK) T cells, and group 3 innate lymphoid cells (ILC3) to secrete proinflammatory cytokines including IL-17, IFN-g, and TNF-a. Risankizumab is the first selective IL-23 antagonist approved for moderate-severe CD. However, there is currently no biomarker to predict response to anti-IL-23 antibodies in IBD. Here, we characterized the cell type- and cytokine-specific pathways in IL-23-responsive cells that predict response to risankizumab in CD patients. Methods Adult patients with a history of ileal, colonic, or ileocolonic CD were included. Active CD in terminal ileum and/or colon were confirmed by colonoscopy and/or MRI. Blood samples were collected within 3 months before and 4, 8, 12, and 52 weeks after the initiation of risankizumab. Peripheral blood mononuclear cells (PBMC) were isolated and cryopreserved for batch analysis using mass cytometry (CyTOF). Pre-treatment PBMCs were stimulated ex vivo with IL-23, and stained for lineage markers to identify Th17, Tc17, gd T, NKT cells, and ILC3, as well as intracellular cytokines including IL-17, IFN-g, TNF-a, IL-22, IL-6, and GM-CSF, using a pre-optimized and validated antibody panel. The CyTOF data were analyzed using the viSNE tool in Cytobank and FlowJo. Patients’ responses were evaluated 12-40 weeks after the initiation of risankizumab based on clinical symptoms and endoscopic/radiographic assessment. Results Among IL-23-responsive cells, increased frequency of NKT cells was identified in the peripheral blood of non-responders before initiation of risankizumab (Figure 1A). In non-responders, Tc17 cells, gd T cells, and ILC3 demonstrated significant elevations in TNF-a, a key pathogenic mediator in CD. gd T cells from non-responders also showed increased IL-17F before treatment. In contrast, NKT cells from non-responders expressed significantly lower GM-CSF, which was previously shown to alleviate intestinal inflammation in patients and mouse models (Figure 1B). Ongoing study is investigating the cytokine production in IL-23-responsive cells after the initiation of risankizumab, as well as before and after starting ustekinumab, an anti-IL-12/IL-23 antibody. Conclusion High dimensional profiling of pre-treatment PBMCs revealed molecular signatures, e.g., elevated TNF-a in multiple IL-23-responsive cells, which are associated with resistance to risankizumab in CD patients. Those findings may help identify response biomarkers for IL-23 antagonists and provide a rationale for using anti-IL-23/anti-TNF combination therapy in some patients with refractory CD.

Abstract P4-08-19: Biomarker analysis: Multi-omics elucidation of Cohort 1 from a phase II study of a triple combination of Atezolizumab + cobimetinib + eribulin in patients with metastatic inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare but aggressive tumor type accounting for up to 10% of breast cancer deaths. One-third of IBCs express high PD-L1 that can be targeted by atezolizumab (Az). MEK inhibitor cobimetinib (Co) not only inhibits the RAS-MAPK pathway but can further enhance immune-mediated killing. Thus, we hypothesize that Az+Co may enhance the efficacy of chemotherapy in metastatic IBC (mIBC). We opened a trial to test this hypothesis with a comprehensive multi-omics biomarker assessment. Patients and Methods: In a single-center, open-label phase II study, cohort 1 received one cycle of Az+ Co, followed by four cycles of Az+Co+eribulin (E) to induce a maximum clinical response, followed by Az+Co maintenance. Pre and Post one cycle of Az+Co tumors were collected for immunohistochemistry (IHC), multiplex immunofluorescence (mIF), whole-exome sequencing (WES), and RNA sequencing (RNAseq). Blood was collected for circulating tumor DNA (ctDNA). Results: Seventeen patients were enrolled in cohort 1. Seven had PR, and three had SD as the best responses. Fourteen had pre, and six had pre/post tumors. The levels of PD-L1 expression at pre/post were not associated with responses. WES revealed the median tumor mutation burden at pre- was 9mt/Mb. More than 50% had TP53 and PI3K pathway mutations at pre. RTK-RAS and Notch pathways were altered in 4/9 cases. PRDM9 and DPY19L2 single-gene mutations were commonly noted in pre. No cancer-associated gene aberration, including potential biomarkers of anti-PDL1 agent response was associated with clinical outcomes. Transcriptomic gene set enrichment analysis demonstrated a greater degree of TNFa and TGFb signaling, Oxphos, angiogenesis, and epithelial-to-mesenchymal transition (EMT) processes in tumors from patients with poor response. Immune profiling by RNAseq revealed two responders to have elevated effector memory T cells, NK T cells, myeloid dendritic cells, and M1 macrophage signatures in pre-samples, but post-samples were not available. mIF confirmed a higher frequency of NK-T cells. The ctDNA analysis from serially collected blood samples is ongoing. Discussion: In this comprehensive multi-omics analysis of pre-and-post-Az+Co, we observed several novel findings, while conventional biomarkers for Az and Co did not correlate with clinical responses. EMT, Oxphos, Notch, and chronic inflammation pathways, which are not previously well reported, were observed in this IBC cohort. These markers warrant further validation to see if they carry significance as therapeutic targets in IBC. Citation Format: Bora Lim, Angela Alexander, Jie S. Willey, Huiming Sun, Suyu Liu, Anisha B. Patel, Edwin Roger Parra, Cara Haymaker, Luisa Solis Soto, Alejandra Serrano, Baohua Sun, Cibelle Freitas Pinto Lima, Auriole Tamegnon, Renganayaki K. Pandurengan, Dzifa Douse, Jessica Lan, Luthra Raja, Randy Chu, Mark Knafl, Scott E. Woodman, Haifeng Zhu, Katja Shulze, Katherine Fedenko, Walter Darbonne, Naoto T. Ueno, Vicente Valero. Biomarker analysis: Multi-omics elucidation of Cohort 1 from a phase II study of a triple combination of Atezolizumab + cobimetinib + eribulin in patients with metastatic inflammatory breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-19.

Remodeling the bladder tumor immune microenvironment by mycobacterial species with changes in their cell envelope composition.

Intravesical BCG instillation after bladder tumor resection is the standard treatment for non-muscle invasive bladder cancer; however, it is not always effective and frequently has undesirable side effects. Therefore, new strategies that improve the clinical management of patients are urgently needed. This study aimed to comprehensively evaluate the bladder tumor immune microenvironment profile after intravesical treatment with a panel of mycobacteria with variation in their cell envelope composition and its impact on survival using an orthotopic murine model to identify more effective and safer therapeutic strategies. tumor-bearing mice were intravesically treated with a panel of BCG and M. brumae cultured under different conditions. Untreated tumor-bearing mice and healthy mice were also included as controls. After mycobacterial treatments, the infiltrating immune cell populations in the bladder were analysed by flow cytometry. We provide evidence that mycobacterial treatment triggered a strong immune infiltration into the bladder, with BCG inducing higher global absolute infiltration than M. brumae. The induced global immune microenvironment was strikingly different between the two mycobacterial species, affecting both innate and adaptive immunity. Compared with M. brumae, BCG treated mice exhibited a more robust infiltration of CD4+ and CD8+ T-cells skewed toward an effector memory phenotype, with higher frequencies of NKT cells, neutrophils/gMDSCs and monocytes, especially the inflammatory subset, and higher CD4+ TEM/CD4+ Treg and CD8+ TEM/CD4+ Treg ratios. Conversely, M. brumae treatment triggered higher proportions of total activated immune cells and activated CD4+ and CD8+ TEM cells and lower ratios of CD4+ TEM cells/CD4+ Tregs, CD8+ TEM cells/CD4+ Tregs and inflammatory/reparative monocytes. Notably, the mycobacterial cell envelope composition in M. brumae had a strong impact on the immune microenvironment, shaping the B and myeloid cell compartment and T-cell maturation profile and thus improving survival. Overall, we demonstrate that the bladder immune microenvironment induced by mycobacterial treatment is species specific and shaped by mycobacterial cell envelope composition. Therefore, the global bladder immune microenvironment can be remodelled, improving the quality of infiltrating immune cells, the balance between inflammatory and regulatory/suppressive responses and increasing survival.

Rosuvastatin Intervention Decreased the Frequencies of the TIM-3+ Population of NK Cells and NKT Cells among Patients with Chronic Hepatitis B.

Natural killer (NK) cells are dichotomously involved in chronic hepatitis B (CHB) infection as principal members of innate immunity. An effective treatment should enhance the antiviral potentials of NK cells and not their immunomodulatory roles. TIM-3 (T-cell immunoglobulin and mucin-containing domain) is a molecule with an essential role in controlling immune tolerance. TIM-3 demonstrated the highest expression among NK cells of patients with chronic liver disorders. Statins have been reported to attenuate the levels of TIM-3 on NK cells. To investigate the frequencies of NK cells, NKT cells, and TIM-3+ population in patients with CHB upon rosuvastatin (RSV) intervention. Thirty confirmed patients with CHB were randomly assigned into two groups of 15 (receiving 20 mg of RSV or placebo per day) for 12 weeks. We evaluated the percentages of TIM-3+ cells by staining the peripheral blood mononuclear cells (PBMCs) with CD3, CD16, and CD56 markers using flow cytometry. Our findings indicated that RSV administration could increase CD3- CD56+ NK cells (P>0.05) and CD3+ CD16+ CD56+ NKT cells (P<0.05). RSV intervention could reduce the percentages of TIM-3+ cells among NK cells (P<0.01) and NKT cells (P> 0.05) of patients with CHB compared with the placebo group. The increased population of NK and NKT cells and the effective reduction of TIM-3+ cells among patients with CHB delineated that rosuvastatin could be proposed as an appropriate modulator of innate immune response (regarding NK and NKT cells) in favor of enhancing their antiviral activities.

Long-term caloric restriction ameliorates T cell immunosenescence in mice

Aging is associated with a decrease in the function of the immune system, a phenomenon known as immunosenescence, which results in reduced resistance to infection. Caloric restriction (CR) is known to prolong lifespan and to regulate immune function. However, whether and how CR affects immunosenescence remains unclear. Here, we evaluated the effect of long- and short-term CR on immunosenescence by subjecting wild-type mice to CR between 6 and 18 months of age or between 17 and 18 months of age, respectively. Compared with a normal diet or short-term CR, long-term CR induced marked or complete attenuation of age-related decreases in the frequency of spleen NK cells and NKT cells; naïve CD4+ and CD8+ T cells; and cytokine- and granzyme B-secreting T cells. In contrast, both long- and short-term CR significantly suppressed age-related upregulation of the T cell exhaustion markers PD-1, Tim-3, and KLRG1, as well as the transcription factors NR4A1 and TOX, which regulate the expression of genes associated with the T cell exhaustion phenotype. These results suggest that CR might suppress age-associated immunosenescence by regulating the expression of transcription factors and target genes that control T cell exhaustion.

Evaluation of Natural Killer Cells in HCV Patients undergoing Hemodialysis

ABSTRACT Background Hepatitis C virus (HCV) is a hepatotropic virus and one of the major causes of liver disease and a potential cause of substantial morbidity and mortality worldwide. Patients on hemodialysis are at high risk for HCV, with frequency of infection several times higher than that in non-uremic patients. Natural killer (NK) cells are best appreciated for innate defense against viral infections and in tumor cell surveillance. Objective To study the percentage of peripheral blood natural killer cells among chronic hepatitis C patients undergoing hemodialysis compared to chronic HCV patients with normal kidney functionsby flowcytometry immunophenotyping. Methodology Frequency of CD3- CD56+ cells was assessed in two distinct groups. Group I: 35 chronic HCV patients with end stage renal disease and undergoing hemodialysis, group II: 35 age and sex matched chronic HCV patients with no kidney disease. Results No significant difference was observed between HCV hemodialysis patients with ESRD and HCV patients with normal kidney function regarding the frequency of both NK cells (CD3CD56+) and NKT cells (CD3+CD56+). However, group I had significantly higher percentage of CD3+ cells than group II. Conclusion no difference was found in this study between HCV patients on hemodialysis and HCV with normal kidney function regarding NK cell frequency. However, assessment of NK cell function in future studies might reveal differences.

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia

SummaryImmune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients’ HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

LEF1 activates a central memory-like program in human NKT cells

Abstract Vα24-invariant natural killer T cells (NKTs) possess potent antitumor activity that can be exploited for use in cancer immunotherapy. We recently demonstrated that the CD62L+ NKT subset persists longer and mediates more potent antitumor activity in vivo than its CD62L− counterpart; as such, CD62L+ NKTs are crucial to maximizing the therapeutic efficacy of NKTs. Key factors involved in transcriptional control of the CD62L+ central memory-like program in NKTs remain largely undefined. To address this gap, we performed comparative gene expression analysis in CD62L+ and CD62L− NKTs and found that LEF1, which encodes a mediator of Wnt/β-catenin signaling, was the top overexpressed gene in the positive subset. We also found that LEF1 protein expression was largely restricted to CD62L+ primary human NKTs and was associated with increased expression of Wnt target genes. While CRISPR-Cas9-mediated knockout of LEF1 expression did not impact CD62L+ cell numbers after initial ex vivo expansion, it did significantly reduce the frequency of these cells after antigenic restimulation. On the other hand, treatment with Wnt/β-catenin signaling activator TWS119 or Wnt3a ligand increased CD62L+ NKT cell frequency. Combined treatment of TWS119 with IL-21, which selectively protects the CD62L+ subset from apoptosis, resulted in significantly higher CD62L+ cell numbers than each single agent alone. These results identify LEF1 as a key transcriptional activator of the central memory-like program in CD62L+ NKTs and pave the way for targeted pharmacological enhancement of NKT cell-based cancer immunotherapy.

Diammonium Glycyrrhizinate Mitigates Liver Injury Via Inhibiting Proliferation Of NKT Cells And Promoting Proliferation Of Tregs.

PurposeDiammonium glycyrrhizinate (DG) is a replacement for glycyrrhizic acid, which is used as a hepatic protector in clinical practice for most liver diseases. The potential role of immune response during autoimmune hepatitis—induced by concanavalin A (Con A)—remains to be elucidated.MethodsC57BL/6J mice were treated with two different doses of DG (75 and 200 mg/kg) 2 hrs before administering Con A. The mice were sacrificed after administering Con A for 0, 6, and 24 hrs. Liver damage grade and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin levels were evaluated. The expression level of cleaved-caspase 3 in liver was detected by Western blotting. Inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interferon γ (IFN-γ) in liver were detected by RT-PCR. Thymus, peripheral blood, spleen, and liver tissues were collected to analyze the percentages of NKT cells, subsets of CD4+CD25−CD69+ and CD8+CD69+ T cells, and subsets of regulatory T cells (Tregs).ResultsOur results revealed that DG pre-treatment significantly decreased the serum ALT and AST levels and improved the histological damage in Con A-induced autoimmune liver injury. Pre-treatment with DG down-regulated the inflammatory cytokines upon challenge with Con A. The DG pre-treatment inhibited the apoptosis of T lymphocytes in the thymus. Further, it effectively suppressed the proliferation of CD4+CD25−CD69+ and CD8+CD69+ subsets in the peripheral blood and spleen. In addition, the DG pretreatment significantly downregulated the frequency of NKT cells, while upregulating the frequency of Tregs in the liver.ConclusionWe believe that the potential protective effect of DG against Con A-induced hepatitis may be partially attributed to its inhibitory activities on inflammatory cytokines in the livers, lymphocyte apoptosis in the thymus, NKT cells proliferation, and activation of CD8+T cells; further, there may also be a possibility of DC promoting Tregs proliferation.

Abstract A130: Metastatic melanoma patients responding to PD1 therapy have higher proportion of peripheral blood NKT-cells

Abstract Anti-PD1 therapy has proven to be effective in various cancer types, but not all patients benefit from the therapy. Further, no comprehensive immunologic monitoring during anti-PD1 treatment has yet been published. In this study, we aimed to discover the effects of anti-PD1 therapy on the immune system, especially on NK and NKT-cells, which are less studied, but known to be involved in antitumor immune events. Peripheral blood samples from immuno-oncology (IO) naive metastatic melanoma patients (n=20) were obtained before the first infusion of pembrolizumab or nivolumab (D0), then 1 and 3 months after the initiation of treatment. From each time-point, complete blood counts (CBC) were obtained, and comprehensive immunophenotyping of NK, NKT, and T-cells was performed with multicolor flow cytometry. Moreover, 92 different serum cytokines were measured using the Olink inflammation panel. The protein levels are presented as arbitrary units of normalized protein expression, NPX, on Log2 scale. The CBC revealed that the proportion of lymphocytes (mean D0 30.6% vs. 3mo 24.9%, p=0.02), decreased during the treatment but no changes were observed in absolute numbers or in other leukocytes. Immunophenotyping of lymphocyte subpopulations revealed that the frequency of NKT brighT-cells was increased (D0 1.8% vs. 1mo 2.3%, p=0.01) and that cytotoxic NK CD56dim cells expressed more CD25 (D0 19.5% vs. 1mo 23.7%, p=0.02) and CD45RO (D0 14.7% vs. 1mo 21.1%, p=0.03) surface markers after 1 month of therapy. The cytokine assay indicated that during anti-PD1 treatment the levels of CXC family cytokines were increased in the serum; CXCL9 (D0 470 vs. 1mo 1070, p=0.0003, D0 470 vs. 3mo 1227, p=0.0007), CXCL11 (D0 49.8 vs. 1mo 81.8, p=0.005), CXCL10 (D0 1000 vs. 1mo 2078, p=0.0003). Also, an increase in IL-12B (D0 31.6 vs. 1mo 41.2, p=0.003, D0 31.6 vs. 3mo 32.4, p=0.04) and TNFRSF9 (D0 126.4 vs. 1mo 181.0, p=0.01, D0 126.4 vs. 3mo 149.0, p=0.04) levels was observed.To further examine these results, patients were categorized into two cohorts: responders (R, n=6, PFS=17.0 months) and patients with progressive disease (PD, n=9, PFS=5.0 months) in terms of the duration of progression-free survival (PFS) and decrease in tumor burden. 5 patients were excluded due to challenging clinical evaluation of response. When examining the differences between these cohorts, CBC indicated a significant decrease in the mean frequency of lymphocytes in PD (D0 26.9% vs. 3mo 19.2%, p=0.04), but not in the R cohort. The responders had also higher frequency of lymphocytes at 1- and 3-month time-points (R 34.5% vs. PD 25.4%, p=0.02, R 32.4% vs. PD 19.2%, p=0.01, respectively) and lower frequency of neutrophils before initiation and after 1 and 3 months of treatment (R 50.8% vs. PD 58.6%, p=0.04, R 45.3% vs. PD 59.4%, p=0.01, R 49.8% vs. PD 64.5%, p=0.04, respectively). The CBC absolute counts revealed that the responders had less neutrophils (R 2.8 109/L vs. PD 4.9 109/L, p=0.04) and monocytes (R 0.4 109/L vs. PD 0.7 109/L, p=0.04) after 3 months of treatment when compared to PD. The immunophenotyping showed that responders had more NKT dim cells before (R 10.1% vs. PD 3.5%, p=0.03) and after 3 months of therapy (R 15.7% vs. PD 3.7%, p=0.03) and the increase in NKT bright frequency was observed only in R (D0 2.5% vs. 1mo 3.4%, p=0.04) and not in PD cohort. The cytokine assay indicated that the CXCL9 was increased in R cohort (D0 476.8 vs. 1mo 1480.2, p=0.01), but not in PD, making the cytokine levels greater in R (R 1480.2 vs. PD 639.3, p=0.01) after 1 month of therapy. Based on our preliminary results, we believe that high frequency of NKT-cells in blood is related to positive treatment response, and in addition to T-cells, also NK and NKT-cells may play a key role in the antitumor response induced by PD-1 inhibition. Hence, further research on the effect of anti-PD1 therapy on NK and NKT-cells is needed to better understand their role in positive therapy response. Citation Format: Henna H.E. Hakanen, Micaela Hernberg, Siru Mäkelä, Bhagwan Yadav, Oscar Brück, Susanna Juteau, Laura Kohtamäki, Mette Ilander, Satu Mustjoki, Kreutzman Anna. Metastatic melanoma patients responding to PD1 therapy have higher proportion of peripheral blood NKT-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A130.

Higher Frequencies of Lymphocytes Expressing the Natural Killer Group 2D Receptor in Patients With Behçet Disease.

Behçet disease (BD) is an inflammatory systemic disease with a fluctuating course, which can affect the skin, eyes, central nervous system, musculoskeletal, gastrointestinal, and vascular systems. No laboratory tests are currently available for the diagnosis of BD and monitoring disease activity. Moreover there is a lack of knowledge on BD pathogenesis. This study focused on circulating Natural Killer (NK), NKT and T cells evaluated as CD3neg CD56pos, CD3pos CD56pos, and CD3pos CD56neg. Peripheral blood mononuclear cells (PBMCs) were collected from 38 BD patients and 20 healthy controls (HC). The frequencies of NK, NKT, and T cells expressing CD16, CD69, NKG2D, Nkp30, Nkp46, and NKG2A were assessed by flow cytometry. Cytotoxic potential of NK cells was evaluated by flow cytometry as the percentage of cells expressing the degranulation marker CD107a after incubation with K562 cells. The levels of 27 cytokines were determined in plasma with a multiplex bead-based assay. Higher percentages of NK, NKT, and T cells expressing NKG2D were detected in PBMCs of BD patients than HC. ROC curve analysis showed that the evaluation of NKG2Dpos NK, NKT, and T cell percentages discriminated between BD patients and HC. Moreover, there was a positive correlation between the BD Current Activity Form (BDCAF) scores and the frequencies of NKG2Dpos NK and NKT cells. A higher frequency of NK cells expressing CD107a was induced in PBMCs from BD patients than HC after incubation with K562 cells. Concentrations of IL-5, IL-6, IL-10, IL-13, IP-10, and MIP-1β were higher in plasma of BD patients than HC. Monitoring the frequencies of NKG2Dpos lymphocytes could help the clinicians in BD patients management. In addition, the increased expression of NKG2D in BD patients is likely involved in disease pathogenesis.

Impaired natural killer cell subset phenotypes in human obesity

Obesity is associated with alterations in functionality of immune cells, like macrophages and natural killer (NK) cells, leading to an increased risk for severe infections and several cancer types. This study aimed to examine immune cell populations and functional NK cell parameters focusing on NK cell subset phenotypes in normal-weight and obese humans. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from normal-weight and obese individuals and analyzed by flow cytometry. Results show no significant changes in the frequency of monocytes, B lymphocytes, or NKT cells but a significantly increased frequency of T lymphocytes in obesity. The frequency of total NK cells was unaltered, whereas the number of low cytotoxic CD56bright NK cell subset was increased, and the number of high cytotoxic CD56dim NK cell subset was decreased in obese subjects. In addition, the frequency of CD56bright NK cells expressing the activating NK cell receptor NKG2D as well as intracellular interferon (IFN)-γ was elevated in the obese study group. In contrast, the frequency of NKG2D- and IFN-γ-positive CD56dim NK cells was lower in obesity compared to normal-weight individuals. Moreover, the expression of the activation marker CD69 was decreased in NK cells, which can be attributed to a reduction of CD69-positive CD56dim NK cells in obese subjects. In conclusion, data reveal an impaired NK cell phenotype and NK cell subset alterations in obese individuals. This NK cell dysfunction might be one link to the higher cancer risk and the elevated susceptibility for viral infections in obesity.

T-lymphocyte-expressing inflammatory cytokines underlie persistence of proteinuria in children with idiopathic nephrotic syndrome

ObjectiveThere is evidence of an important role of immune system changes in the triggering and maintenance of idiopathic nephrotic syndrome (INS). The aim of this study was to investigate the expression of cytokines in lymphocyte populations of patients with INS in comparison to healthy individuals, according to proteinuria. MethodsThis cross-sectional study included 44 patients with INS and eight healthy children, matched for age and sex (controls). Patients were subdivided according to proteinuria: persistent proteinuria or partial remission (PP≥300mg/24h, n=17) and low proteinuria or complete remission (LP<300mg/24h, n=27). Ex vivo analysis of peripheral blood leukocytes by flow cytometry was performed using surface markers for T-lymphocytes, TCD4, TCD8, natural killer (NK) cells, NKT, and B-lymphocytes. Frequencies of intracellular cytokines were analyzed in these cells. ResultsThe frequencies of B-lymphocytes, NK cells, and NKT cells were lower in INS than in controls, whereas INS patients had a higher frequency of CD4+tumor necrosis factor (TNF)-α+ cells than controls. Cytotoxic-T-lymphocytes expressing IFN-γ were lower in INS than in controls. Patients with PP showed higher frequencies of CD4-T-lymphocytes expressing IFN-γ and TNF-α than controls. CD8-lymphocytes expressing TNF-α were increased in PP group when compared with LP and controls, while CD8+interferon (IFN)-γ+ cells were lower than in LP and in controls. ConclusionRegardless the level of proteinuria, INS patients had increased expression of TNF-α in CD4-lymphocytes and reduced expression of IFN-γ in CD8-lymphocytes. Persistence of proteinuria was associated with higher levels of inflammatory markers.

Basic Science Liver.

Basic Science Liver.

Fatty acid amide hydrolase (FAAH) blockade ameliorates experimental colitis by altering microRNA expression and suppressing inflammation

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), which is thought to result from immune-mediated inflammatory disorders, leads to high morbidity and health care cost. Fatty acid amide hydrolase (FAAH) is an enzyme crucially involved in the modulation of intestinal physiology through anandamide (AEA) and other endocannabinoids. Here we examined the effects of an FAAH inhibitor (FAAH-II), on dextran sodium sulphate (DSS)-induced experimental colitis in mice. Treatments with FAAH-II improved overall clinical scores by reversing weight loss and colitis-associated pathogenesis. The frequencies of activated CD4+ T cells in spleens, mesenteric lymph nodes (MLNs), Peyer’s patches (PPs), and colon lamina propiria (LP) were reduced by FAAH inhibition. Similarly, the frequencies of macrophages, neutrophils, natural killer (NK), and NKT cells in the PPs and LP of mice with colitis declined after FAAH blockade, as did concentrations of systemic and colon inflammatory cytokines. Microarray analysis showed that 26 miRNAs from MLNs and 217 from PPs had a 1.5-fold greater difference in expression after FAAH inhibition. Among them, 8 miRNAs were determined by reverse-transcription polymerase chain reaction (RT-PCR) analysis to have anti-inflammatory properties. Pathway analysis demonstrated that differentially regulated miRNAs target mRNA associated with inflammation. Thus, FAAH-II ameliorates experimental colitis by reducing not only the number of activated T cells but also the frequency of macrophages, neutrophils, and NK/NKT cell, as well as inflammatory miRNAs and cytokine at effector sites in the colon. These studies demonstrate for the first time that FAAH-II inhibitor may suppress colitis through regulation of pro-inflammatory miRNAs expression.

Improving the immunogenicity of dendritic cell-derived exosome-based vaccines for the immunotherapy of melanoma

Abstract Dendritic cell (DC)-derived exosomes (Dexo) are promising vaccine candidates for the treatment of cancer. We aimed at identifying a Dexo formulation with stronger immune stimulatory properties and efficacy as compared to the Dexo vaccines previously tested in pre-clinical and clinical settings of cancer immunotherapy. We therefore produced Dexo from DCs loaded with specific antigens and matured with the Toll-like receptor (TLR)-3 ligand poly(I:C) as danger signal. In vivo, Dexo produced from DCs loaded with OVA and stimulated with poly(I:C) could induce efficient activation of endogenous OVA-specific CD4+ and CD8+ T cells, leading to pro-inflammatory Th1 immune responses. We subsequently produced Dexo from DCs matured with poly(I:C) and cultured in the presence of oxidized B16F10 cell lysates (Dexo(B16+pIC)) as a source of B16F10 antigens. Mice bearing subcutaneous B16F10 tumors were vaccinated intradermally to target tumor-draining lymph nodes (tdLN), resulting in reduced tumor growth, limited metastasis burden and, most importantly, in prolonged survival of the mice vaccinated with Dexo(B16+pIC). Such benefits were associated with increased frequencies of effector CD8+ T cells in the tdLNs, spleen and tumor masses, reduced frequencies of tumor-infiltrating exhausted PD-1+ CD8+ T cells and increased frequencies of tumor-infiltrating NK and NK-T cells obtained upon vaccination with Dexo(B16+pIC) as compared to Dexo(B16), which instead are devoid of poly(I:C) and resemble the Dexo vaccines previously tested in human patients. Our results demonstrate that poly(I:C) is a promising candidate for the production of Dexo vaccines with improved immune stimulatory properties suitable for the immunotherapy of cancer.

Abstract A09: Improving the immunogenicity of dendritic cell-derived exosome vaccines for the immunotherapy of melanoma

Abstract Dendritic cell (DC)-derived exosomes (Dexo) are promising vaccine candidates since they carry key molecules, normally found in DCs, that are essential for the activation of adaptive immune responses. Moreover, Dexo have a longer in vivo half-life than DCs, are not subject to the effects of immunomodulators, and are devoid of the risks of cell-based vaccines. As such, clinical trials were conducted to test the feasibility, safety and efficacy of vaccines composed of Dexo carrying tumor antigens for the immunotherapy of melanoma and small-cell lung carcinoma patients. Even though these trials proved the feasibility and safety of Dexo vaccination for the treatment of human cancer patients, the clinical benefits associated with the treatment were limited. We reasoned that the limited efficacy of Dexo vaccines could be ascribed to the low immunogenicity of the Dexo formulations tested in the clinical trials. Indeed, such formulations were produced from DCs loaded with tumor antigens in the absence of any pro-inflammatory stimuli that could provide the danger signals necessary to elicit a proper anti-tumor immune response in vivo. The objective of our work was thus to identify a Dexo formulation with stronger immune stimulatory properties as compared to the Dexo formulations previously tested in pre-clinical and clinical settings. As adaptive immune responses are elicited by antigen-presenting cells activated in the presence of antigens and pro-inflammatory signals, we produced Dexo from DCs loaded with specific antigens (either Ovalbumin, OVA, or tumor-derived antigens) and matured with the Toll-like receptor (TLR)-3 ligand poly(I:C) as danger signal. As a preliminary assessment, we tested Dexo produced from DCs loaded with OVA and stimulated with poly(I:C) (Dexo(OVA+pIC) in in vivo models of vaccination, proving that Dexo(OVA+pIC) could induce efficient activation of transferred or endogenous OVA-specific CD4+ and CD8+ T cells, leading to pro-inflammatory Th1 immune responses. We then moved to a model of therapeutic vaccination in melanoma tumor-bearing mice, taking advantage of the B16F10 melanoma model. To this purpose, we produced Dexo from DCs matured with poly(I:C) and cultured in the presence of oxidized B16F10 cell lysates (Dexo(B16+pIC)), in order to provide additional pro-inflammatory stimuli as well as all the B16F10 antigens to the exosome-producing DCs. Mice bearing subcutaneous B16F10 tumors were vaccinated intradermally in order to target tumor-draining lymph nodes (tdLN). This protocol of vaccination resulted in reduced tumor growth and limited metastasis burden and, most importantly, in prolonged survival of the mice vaccinated with Dexo(B16+pIC). Such benefits were associated with increased frequencies of effector CD8+ T cells in the tdLNs, spleen and tumor masses, reduced frequencies of tumor-infiltrating exhausted PD-1+ CD8+ T cells and increased frequencies of tumor-infiltrating NK and NK-T cells obtained upon vaccination with Dexo(B16+pIC) as compared to vaccination with Dexo(B16). Our results demonstrate that poly(I:C) is a promising candidate for the production of Dexo vaccines with improved immune stimulatory properties suitable for the immunotherapy of cancer. Citation Format: Martina Damo, David Scott Wilson, Jeffrey Alan Hubbell. Improving the immunogenicity of dendritic cell-derived exosome vaccines for the immunotherapy of melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A09.

Diabetes Mellitus Directs NKT Cells Toward Type 2 and Regulatory Phenotype / Diabetes Melitus Usmerava Diferencijaciju NKT Celija U Pravcu Tip 2 I Regulatornog Fenotipa

Abstract Diabetes mellitus is chronic disorder characterized by hyperglycaemia. Hyperglycaemia induces mitochondrial dysfunction, enhances oxidative stress and thus promotes reactive oxygen species (ROS) production. Earlier studies suggested that reactive oxygen species (ROS) are involved in the pathogenesis of many diseases. Previous studies have revealed that hyperglycaemia changes the functional phenotype of monocytes, macrophages, neutrophils, NK cells and CD8+ T cells. The aim of this study was to investigate whether diabetes affects the functional phenotype of NKT cells. Diabetes mellitus was induced in BALB/c mice by intraperitoneal injection of streptozotocin at a single dose of 170 mg/kg body weight. The number and functional phenotype of splenic NKT cells was assessed by fl ow cytometry, 28 days after diabetes induction. The diabetic condition facilitated the production of antioxidant enzymes, including catalase (p&lt;0.05) and superoxide dismutase. Hyperglycaemia enhanced oxidative stress and thus decreased the number of splenic NKT cells but did not change the percentage of splenic CD3+CD49+ NKT cells that express the activatory receptor NKP46 or produce IFN-γ. However, hyperglycaemia increased the frequency of splenic NKT cells that express KLRG-1 and produce TGF-β, IL-4, and IL-5, and it decreased the frequency of IL-17+ NKT cells. Our study indicates that diabetes mellitus induces oxidative stress and switches the functional phenotype of NKT cells towards type 2 (IL-4 and IL-5 producing NKTs) and regulatory (TGF-β Thproducing NKTs) phenotypes. These findings are correlated with the clinical observation in humans that diabetic patients are more prone to infections and tumours.

Blocking fatty acid amide hydrolase reduces T cell activation and attenuates experimental colitis (IRC10P.405)

Abstract Inflammatory bowel disease (IBD) refers to chronic inflammatory disorders of the gut with unknown etiology. Fatty acid amide hydrolase (FAAH) is the enzyme crucially involved in the modulation of intestinal physiology. We investigated the effects of FAAH inhibitors on the dextran sodium sulfate (DSS) induced model of colitis. FAAH inhibitors diminished the overall clinical score, reversed the colitis-associated inflammation score, and reversed body weight loss after DSS induced colitis. The frequency of activated CD4+ T cells in the spleen, mesenteric lymph node (MLN), and Peyer’s patches (PP) was reduced after FAAH inhibition as compared to the control group. Further, the frequency of macrophages, natural killer (NK1.1), and NKT cells in PP, and the concentration of inflammatory cytokines and chemokines also declined after FAAH inhibition. FAAH treatment also resulted in greater than 1.5 fold changes in 26 miRs in MLN and 217 miRNAs in PP. Among these, eight anti-inflammatory miRNAs were validated by RT-PCR analysis. Pathway analysis also validated that differentially regulated miRNAs target mRNA associated with inflammation. Taken together, these results indicate that FAAH modulates colitis severity by reducing activated T cells at sites of inflammation and suppresses other immune mediators such as systemic cytokines and inflammatory miRNAs through regulation of miRs. In summary, our results suggest that FAAH inhibitors may be developed as a novel therapeutic agent for IBD.