Inducer Cells Research Articles

Clinical and Experimental Study of High Mobility Group Box-2 and Valvular Calcification in Elderly Patients with Degenerative Heart Valve Disease

Introduction: The aim of this study was to investigate the relationship between the high mobility group box-2 (HMGB2) and valve calcification in senile degenerative heart valve disease (SDHVD). Methods: According to the echocardiographic results, patients with calcified heart valves were used as the experimental group and patients without calcified heart valves were used as the control group; blood was drawn for testing, and serum levels of HMGB2 were measured by an enzyme-linked immunosorbent assay. Human heart valve interstitial cells (hVICs) cultured in vitro were randomly divided into two groups. The calcification group was cultured with a medium containing calcification induction solution and cells were induced on days 1, 3, and 5, and the control group was cultured with a standard medium. Expression of bone morphogenetic protein 4 (BMP-4) and HMGB2 in both groups was detected by Western blot. RT-PCR was performed to detect the expression of the HMGB2 gene during calcification. The hVICs were cultured in vitro for 4 days with different concentrations of exogenous HMGB2 (0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 2 μg/mL), while the control group was cultured with a standard medium and the expression of BMP-4 and NF-κB P65 was detected by Western blot. Results: The serum level of HMGB2 was 7.90 (5.92, 12.39) μg/L, higher than that of 7.06 (5.06, 9.73) μg/L in the valve calcification group in elderly patients with degenerative valve disease (p = 0.005); the differences were statistically significant. In in vitro experiments, the cellular calcification protein BMP-4 and the HMGB2 protein were higher in the calcification group compared to the control group (p < 0.05). Exogenous stimulation of hVICs with HMGB2 was able to upregulate the expression of BMP-4 and NF-κB P65 (p < 0.05). Conclusions: HMGB2 is correlated with valvular calcification in senile degenerative heart valve disease. The HMGB2 protein may promote the process of SDHVD valve calcification by activating the NF-κB pathway and upregulating the expression of BMP-4.

Ruminococcus gnavus and Limosilactobacillus reuteri Regulate Reg3γ Expression through Multiple Pathways.

Epithelium-derived antimicrobial peptides represent an evolutionarily ancient defense mechanism against pathogens. Regenerating islet-derived protein 3 γ (Reg3γ), the archetypal intestinal antimicrobial peptide, is critical for maintaining host-microbe interactions. Expression of Reg3γ is known to be regulated by the microbiota through two different pathways, although it remains unknown whether specific Reg3γ-inducing bacteria act via one or both of these pathways. In recent work, we identified Ruminococcus gnavus and Limosilactobacillus reuteri as commensal bacteria able to induce Reg3g expression. In this study, we show these bacteria require myeloid differentiation primary response protein 88 and group 3 innate lymphoid cells for induction of Reg3γ in mice. Interestingly, we find that R. gnavus and L. reuteri suppress Reg3γ in the absence of either myeloid differentiation primary response protein 88 or group 3 innate lymphoid cells. In addition, we demonstrate that colonization by these bacteria is not required for induction of Reg3γ, which occurs several days after transient exposure to the organisms. Taken together, our findings highlight the complex mechanisms underlying microbial regulation of Reg3γ.

ICOS/ICOSLG and PD-1 Co-Expression is Associated with the Progression of Colorectal Precancerous- Carcinoma Immune Microenvironment.

This study aimed to investigate the expression of inducible T-cell co-stimulator (ICOS) and its ligand (ICOSLG), along with their association with clinicopathological features and influence on the immune profile in colorectal cancer (CRC). The Cancer Genome Atlas Colorectal Adenocarcinoma cohorts were used. We also analyzed 131 clinical samples of colon lesions, including precancerous lesions (hyperplastic polyps, low-grade dysplasia, and high-grade dysplasia) and CRC tissues. We conducted immunohistochemical (IHC) assays and multiple IHC (mIHC) of CD4+, Foxp3+ tumor-infiltrating lymphocytes (TILs), and PD-1/PD-L1 immune checkpoints in precancerous lesions and CRC samples from our patient subsets to determine changes and correlations in ICOS and ICOSLG expression during progression through the adenoma-carcinoma pathway. High expression of ICOS and ICOSLG was a significant factor in CRC in multiple analyses and was positively correlated with CD4+/Foxp3+ TIL density and PD-1/PD-L1 expression, which increased with the sequential progression of lesions from precancerous tissues to carcinoma. Multivariable logistic regression analysis suggested that the location and expression level of ICOS/ICOSLG may be involved in precancerous-carcinoma progression. The co-expression status of PD-1 and ICOS/ ICOSLG could stratify patients with colorectal lesions into three groups of low, moderate, and high risk of progression. According to this classification and mIHC assays, we found a strong correlation between increased PD-1+ICOS+ or PD-1+ICOSLG+ co-expression and CRC, which might be deemed an independent factor in carcinogenesis. Increased ICOS/ICOSLG expression may be associated with the progressive formation of Foxp3+ TILs in the immune microenvironment and may further promote the development of the abnormal cytology of colorectal lesions from precancerous neoplasia to CRC. Our findings support the interpretation that enhanced co-expression of PD-1+ICOS+ or PD-1+ICOSLG+ contributes to the immune-active microenvironment of the colorectal adenoma-carcinoma sequence.

Targeting the Inducible T-cell Costimulator (ICOS) in Patients with Relapsed/Refractory T-follicular Helper Phenotype Peripheral T-cell and Angioimmunoblastic T-cell Lymphoma.

Proliferation of T-follicular helper (TFH) CD4+ T cells is a postulated pathogenic mechanism for T-cell non-Hodgkin lymphomas (T-NHL). The inducible T-cell costimulator (ICOS) is highly expressed by TFH, representing a potential target. MEDI-570 is a monoclonal antibody against ICOS, which eliminates ICOS+ cells in preclinical models. We report the safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of MEDI-570 in T-NHL. NCI-9930 is a phase I, first-in-human study of MEDI-570 in relapsed/refractory malignant T-NHL known to express ICOS. MEDI-570 was administered intravenously every 3 weeks for up to 12 cycles. Primary endpoints were safety, dose-limiting toxicities (DLT), and recommended phase II dose (RP2D). Secondary and exploratory endpoints included efficacy parameters and various correlative studies. This study is supported by the National Cancer Institute (NCT02520791). Twenty-three patients were enrolled and received MEDI-570 at five dose levels (0.01-3 mg/kg). Sixteen (70%) had angioimmunoblastic T-cell lymphoma (AITL); median age was 67 years (29-86) and the median prior lines of therapies was 3 (1-16). Most common grade 3 or 4 adverse events were decreased CD4+ T cells (57%), lymphopenia (22%), anemia (13%), and infusion-related reactions (9%). No DLTs were observed. The RP2D was determined at 3 mg/kg. Analysis of T-cell subsets showed reductions in CD4+ICOS+ T cells reflecting its effects on TFH cells. The response rate in AITL was 44%. MEDI-570 was well tolerated and showed promising clinical activity in refractory AITL. MEDI-570 resulted in sustained reduction of ICOS+ T lymphocytes.

P21CIP/WAF1 saRNA inhibits proliferative vitreoretinopathy in a rabbit model.

Proliferative vitreoretinopathy (PVR) is a disease process resulting from proliferation of retinal pigment epithelial (RPE) cells in the vitreous and periretinal area, leading to periretinal membrane formation and traction and eventually to postoperative failure after vitreo-retinal surgery for primary rhegmatogenous retinal detachment (RRD). The present study was designed to test the therapeutic potential of a p21CIP/WAF1 (p21) inducing saRNA for PVR. A chemically modified p21 saRNA (RAG1-40-53) was tested in cultured human RPE cells for p21 induction and for the inhibition of cell proliferation, migration and cell cycle progression. RAG1-40-53 was further conjugated to a cholesterol moiety and tested for pharmacokinetics and pharmacodynamics in rabbit eyes and for therapeutic effects after intravitreal administration in a rabbit PVR model established by injecting human RPE cells. RAG1-40-53 (0.3 mg, 1 mg) significantly induced p21 expression in RPE cells and inhibited cell proliferation, the progression of cell cycle at the G0/G1 phase and TGF-β1 induced migration. After a single intravitreal injection into rabbit eyes, cholesterol-conjugated RAG1-40-53 exhibited sustained concentration in the vitreal humor beyond at least 8 days and prevented the progression of established PVR. p21 saRNA could represent a novel therapeutics for PVR by exerting a antiproliferation and antimigration effect on RPE cells.

Gap and Force Adjustment during Laser Beam Welding by Means of a Closed-Loop Control Utilizing Fixture-Integrated Sensors and Actuators

The development of adaptive and intelligent clamping devices allows for the reduction of rejects and defects based on weld discontinuities in laser-beam welding. The utilization of fixture-integrated sensors and actuators is a new approach, realizing adaptive clamping devices that enable in-process data acquisition and a time-dependent adjustment of process conditions and workpiece position by means of a closed-loop control. The present work focused on sensor and actuator integration for an adaptive clamping device utilized for laser-beam welding in a butt-joint configuration, in which the position and acting forces of the sheets to be welded can be adjusted during the process (studied welding speeds: 1 m/min, 5 m/min). Therefore, a novel clamping system was designed allowing for the integration of inductive probes and force cells for obtaining time-dependent data of the joint gap and resulting forces during welding due to the displacement of the sheets. A novel automation engineering concept allowed the communication between different sensors, actuators and the laser-beam welding setup based on an EtherCAT bus. The subsequent development of a position control and a force control and their combination was operated with a real time PC as master in the bus system and proved the feasibility of the approach based on proportional controllers. Finally, the scalability regarding higher welding speeds was demonstrated.

Microfluidic device for differentiation induction of iPS cells derived embryoid bodies with local chemical stimulation

AbstractThis paper reports a microfluidic device that allows us to measure the effect of local differentiation stimulation on embryoid bodies for the purpose of elucidating the role of the cell‐cell interaction of iPS cells in differentiation induction. We developed the local stimulation device having two chambers separated by thin polydimethyl siloxane (PDMS) membrane with through‐holes of the diameter smaller than single cell. Embryoid bodies of iPS cells are formed on the membrane and seal the holes, separating the aqueous environments of two chambers without leakage. This configuration brings localized exposure to chemicals for differentiation induction at the area defined by the holes. In this paper, we demonstrated the function of local stimulation using fluorescent solution.

Differential Modulation of Human M1 and M2 Macrophage Activity by ICOS-Mediated ICOSL Triggering.

Activated T cells express the inducible T-cell co-stimulator (ICOS) that, upon binding to its ubiquitously expressed ligand (ICOSL), regulates the immune response and tissue repair. We sought to determine the effect of ICOS:ICOSL interaction on human M1 and M2 macrophages. M1 and M2 macrophages were polarized from monocyte-derived macrophages, and the effect of a soluble recombinant form of ICOS (ICOS-CH3) was assessed on cytokine production and cell migration. We show that ICOS-CH3 treatment increased the secretion of CCL3 and CCL4 in resting M1 and M2 cells. In LPS-treated M1 cells, ICOS-CH3 inhibited the secretion of TNF-α, IL-6, IL-10 and CCL4, while it increased that of IL-23. In contrast, M2 cells treated with LPS + IL4 displayed enhanced secretion of IL-6, IL-10, CCL3 and CCL4. In CCL7- or osteopontin-treated M1 cells, ICOS-CH3 boosted the migration rate of M1 cells while it decreased that of M2 cells. Finally, β-Pix expression was upregulated in M1 cells and downregulated in M2 cells by treatment with ICOS-CH3. These findings suggest that ICOSL activation modulates the activity of human M1 and M2 cells, thereby eliciting an overall anti-inflammatory effect consistent with its role in promoting tissue repair.

IRF8 Regulates Intrinsic Ferroptosis through Repressing p53 Expression to Maintain Tumor Cell Sensitivity to Cytotoxic T Lymphocytes.

Ferroptosis has emerged as a cytotoxic T lymphocyte (CTL)-induced tumor cell death pathway. The regulation of tumor cell sensitivity to ferroptosis is incompletely understood. Here, we report that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified the ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells acquire resistance to intrinsic ferroptosis induction and IRF8-deficient tumor cells also exhibit decreased ferroptosis in response to tumor-specific CTLs. Irf8 deletion increased p53 expression in tumor cells and knocking out p53 in IRF8.KO tumor cells restored tumor cell sensitivity to intrinsic ferroptosis induction. Furthermore, IRF8.KO tumor cells grew significantly faster than WT tumor cells in immune-competent mice. To restore IRF8 expression in tumor cells, we designed and synthesized codon usage-optimized IRF8-encoding DNA to generate IRF8-encoding plasmid NTC9385R-mIRF8. Restoring IRF8 expression via a lipid nanoparticle-encapsulated NTC9385R-mIRF8 plasmid therapy suppressed established tumor growth in vivo. In human cancer patients, nivolumab responders have a significantly higher IRF8 expression level in their tumor cells as compared to the non-responders. Our data determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis.

Gasdermin D pore-forming activity is redox-sensitive

Reactive oxygen species (ROS) regulate the activities of inflammasomes, which are innate immune signaling organelles that induce pyroptosis. The mechanisms by which ROS control inflammasome activities are unclear and may be multifaceted. Herein, we report that the protein gasdermin D (GSDMD), which forms membrane pores upon cleavage by inflammasome-associated caspases, is a direct target of ROS. Exogenous and endogenous sources of ROS, and ROS-inducing stimuli that prime cells for pyroptosis induction, promote oligomerization of cleaved GSDMD, leading to membrane rupture and cell death. We find that ROS enhance GSDMD activities through oxidative modification of cysteine 192 (C192). Within macrophages, GSDMD mutants lacking C192 show impaired ability to form membrane pores and induce pyroptosis. Reciprocal mutagenesis studies reveal that C192 is the only cysteine within GSDMD that mediates ROS responsiveness. Cellular redox state is therefore a key determinant of GSDMD activities.

Myeloid-derived suppressor cells promote the formation of abdominal aortic aneurysms through the IL-3-ICOSL-ICOS axis

Th17 cells are powerful inflammation promoters in the pathogenesis of abdominal aortic aneurysms (AAAs). Myeloid-derived suppressor cells (MDSCs) can promote the differentiation of Th17 cells in chronic inflammatory autoimmune injury. Here, we aim to examine whether MDSCs regulate the differentiation of Th17 cells to participate in the development of AAA. We demonstrated an abnormal accumulation of MDSCs in AAA patients, which was positively associated with Th17 cells. We established angiotensin II-induced apolipoprotein E knockout mice and found the impaired immunosuppressive function of M-MDSCs. After systemic injection of anti-Gr-1 antibody in AAA mice to deplete circulating MDSCs, AAA formation and the differentiation of Th17 cells were abolished, and the overexpression of inducible T-cell costimulator (ICOS) on Th17 cells was reversed accordingly. Regulating the expression of ICOS ligand (ICOSL) on MDSCs affects the differentiation of Th17 cells. The adoptive transfer of ICOSLlowMDSCs in AAA mice inhibited the differentiation of Th17 cells and the development of AAA. Meanwhile, rIL-3 promoted the survival and immunosuppressive dysfunction of MDSCs, upregulated ICOSL expression on MDSCs by inhibiting activation of the PI3K/AKT signaling pathway, and regulated MDSCs to promote the differentiation of Th17 cells via the ICOSL-ICOS axis. An increase in serum IL-3, ICOSL+MDSCs, and ICOS+Th17 cells was detected in AAA patients, and IL-3 levels were positively correlated with the proportion of ICOSL+MDSC cells. In conclusion, we uncovered a pivotal role of MDSCs in promoting the differentiation of Th17 cells through the IL-3-ICOSL-ICOS axis during AAA, providing an important theoretical basis for understanding the pathogenesis of AAA.

Experimental Evaluation of the Wet Steam Flow Electrification Effect on Its Dielectric Properties

It has been established that in order to study thermo- and electrophysical phenomena in wet steam turbines, studies of the wet steam flow volume charge effect on its dielectric and thermophysical properties have recently been carried out at A. Pidhornyi Institute of Mechanical Engineering Problems of the National Academy of Sciences of Ukraine. According to their results, it was established that the most representative electrophysical parameter, which allows to evaluate changes in the thermophysical properties of steam, which occur under the action of its own volume charge, is its dielectric constant. It is assumed that the value of the dielectric constant of an electrified steam can be significantly different from the value for a neutral steam, and a mathematical assessment of its possible change is made. It has been confirmed that the influence of electrophysical phenomena caused by the wet steam flow electrification is significant, but is not taken into account in the existing physical and mathematical thermodynamic models of the steam expansion process. It is proved that in order to clarify the main thermodynamic parameters and calculated characteristics of the electrified wet steam flow, it is necessary to determine how its dielectric constant changes. On the basis of the analysis, the relevance of experimental determination of the dielectric constant of a wet steam flow with a volume charge in order to obtain the dependence of its change on the temperature and pressure of the flow, as well as the density of the volume charge, is substantiated. To perform the research, a gas dynamic laboratory plant, which allows to obtain a wet steam flow with a volume charge, was used. The internal space of the flow part of a real wet steam turbine has a significant size and allows the formation of a flow with a volume charge of a complex spatial configuration and structure. It is emphasized that in the flow part of the plant of a small volume, in contrast to the turbine flow part, significant technical difficulties arise when organizing the conditions for the occurrence of a wet steam flow with a volume charge. Taking this into account, at the first stage, it was decided to conduct a study of a steam flow with a volume charge flowing into the atmosphere in a laboratory room with a sufficient volume to form its spatial structure. To estimate the value of its dielectric constant, the inductive method (L-method) of determining dielectric properties, in which the substance under study is introduced into the inductive solenoid cell, was chosen. Experiments were conducted and, according to the obtained data, it is possible to make a preliminary assessment of the change in dielectric constant in the presence of a volume electric charge in the steam flow

Identification of novel NFKB1 and ICOS frameshift variants in patients with CVID.

Common variable immunodeficiency (CVID) is a 'late-onset' primary immunodeficiency characterized by variable manifestations and genetic heterogeneity. A monogenic cause of CVID has been reported in 10% of patients. In this study, we identified two novel pathogenic variants implicated in monogenic CVID by whole exome sequencing (WES) analysis: a heterozygous nuclear factor κB subunit 1 (NFKB1) p.G686fs mutation and a homozygous inducible T-cell co-stimulator (ICOS) p.L96Sfs mutation. The predicted crystal models indicated premature truncation of the two mutated proteins. Both variants were demonstrated as loss-of-function mutations and were associated with overlapped manifestations of respiratory fungal infection and splenomegaly. We further performed a detailed assessment of immunologic phenotypes and impaired lymphocyte functions in patients. Moreover, we discovered an association between monoclonal T-large granular lymphocyte proliferation and ICOS-deficient CVID for the first time. These observations lead to a new perspective on the underlying genetic heterogeneity of CVID.

Circulating plasmablasts and follicular helper T-cell subsets are associated with antibody-positive autoimmune epilepsy.

Autoimmune epilepsy (AE) is an inflammatory disease of the central nervous system with symptoms that have seizures that are refractory to antiepileptic drugs. Since the diagnosis of AE tends to rely on a limited number of anti-neuronal antibody tests, a more comprehensive analysis of the immune background could achieve better diagnostic accuracy. This study aimed to compare the characteristics of anti-neuronal antibody-positive autoimmune epilepsy (AE/Ab(+)) and antibody-negative suspected autoimmune epilepsy (AE/Ab(-)) groups. A total of 23 patients who met the diagnostic criteria for autoimmune encephalitis with seizures and 11 healthy controls (HC) were enrolled. All patients were comprehensively analyzed for anti-neuronal antibodies; 13 patients were identified in the AE/Ab(+) group and 10 in the AE/Ab(-) group. Differences in clinical characteristics, including laboratory and imaging findings, were evaluated between the groups. In addition, the immunophenotype of peripheral blood mononuclear cells (PBMCs) and CSF mononuclear cells, particularly B cells and circulating Tfh (cTfh) subsets, and multiplex assays of serum and CSF were analyzed using flow cytometry. Patients with AE/Ab(+) did not show any differences in clinical parameters compared to patients with AE/Ab(-). However, the frequency of plasmablasts within PBMCs and CSF in patients with AE/Ab(+) was higher than that in patients with AE/Ab(-) and HC, and the frequency of cTfh17 cells and inducible T-cell co-stimulator (ICOS) expressing cTfh17 cells within cTfh subsets was higher than that in patients with AE/Ab(-). Furthermore, the frequency of ICOShighcTfh17 cells was positively correlated with that of the unswitched memory B cells. We also found that IL-12, IL-23, IL-6, IL-17A, and IFN-γ levels were elevated in the serum and IL-17A and IL-6 levels were elevated in the CSF of patients with AE/Ab(+). Our findings indicate that patients with AE/Ab(+) showed increased differentiation of B cells and cTfh subsets associated with antibody production. The elevated frequency of plasmablasts and ICOS expressing cTfh17 shift in PBMCs may be indicative of the presence of antibodies in patients with AE.

Interleukin-13 inhibition by tralokinumab reduces inducible T-cell costimulator-positive innate lymphoid cells in skin lesions of atopic dermatitis.

Despite the low frequency of skin ILCs and the limited number of samples analyzed in this study, our data indicate that ICOS+ ILCs express IL-13Rα1 and that the density of ICOS+ ILCs decreased four weeks after initiation of treatment with tralokinumab.

Efficacy and exploratory biomarker analysis of entinostat plus exemestane in advanced or recurrent breast cancer: phase II randomized controlled trial.

We aimed to confirm the efficacy and safety of the oral histone deacetylase inhibitor entinostat in Japanese patients with hormone receptor-positive advanced/recurrent breast cancer and to explore potential biomarkers. This phase II, double-blind, randomized, placebo-controlled trial (ClinicalTrials.gov; NCT03291886) was conducted at 28 Japanese sites (September 2017-July 2020; interim analysis cutoff: April 2019). Patients with progression/relapse following non-steroidal aromatase inhibitors were randomized 1:1 to entinostat (5mg/week) or placebo, plus exemestane (25mg/day). Primary endpoint was progression-free survival; secondary endpoints included overall survival and safety. Exploratory biomarker outcomes included lysine acetylation, immune cell profiles, estrogen receptor 1 mutations and plasma chemokines. Of 133 randomized patients, 131 (65 entinostat, 66 placebo) who received study drug were analyzed. Median (95% confidence interval) progression-free survival was 5.8 (3.2-7.8) months for entinostat and 3.3 (3.1-5.8) months for placebo (hazard ratio [95% confidence interval]: 0.75 [0.50-1.14]; P=0.189). Median overall survival was not reached in either group. Entinostat tended to prolong progression-free survival in patients aged ≥65years, not endocrine resistant, or with estrogen receptor 1 Y537S mutation. Candidate biomarkers of efficacy (progression-free survival) included lysine acetylation in CD3+ cells, plasma interferon gamma-induced protein 10, dendritic cell CD86 expression, and CD4+ cell expression of human leukocyte antigen-DR and inducible T-cell co-stimulator. Safety was similar to non-Japanese populations; however, seven entinostat-treated patients (10.8%) had reversible lung injury. In Japanese patients, the safety of entinostat plus exemestane was acceptable and progression-free survival was prolonged, although not significantly. Exploratory analyses identified potential biomarkers, including lysine acetylation, of efficacy.

Transcription Factor Fli-1 Regulates T Cell Immunity and Tolerance in Gvh and GVL Responses

The E26 transformation-specific (ETS) transcription factor (TF) family member Friend Leukemia Virus Integration 1 (Fli-1) is linked to the oncogenesis of virally induced leukemia and Ewing's Sarcoma, as well as the development of autoimmune diseases including systemic lupus erythematosus and systemic sclerosis. A recent report presents compelling evidence that Fli-1 restrains CD8 effector T cell differentiation and protective immunity against cancer. How Fli-1 regulates primary T cell differentiation and function remain largely elusive. In this study, we investigated the role of Fli-1 in T-cell pathogenicity of graft-versus-host disease (GVHD) and validated Fli-1 as a therapeutic target. Using conditional knock-out models featured as homozygous (Fli-1KO) or heterozygous (Fli-1HET) deficiency of Fli-1 in T cells, we found that Fli-1 dynamically regulates different T-cell subsets in allogeneic responses and pathogenicity in the development of acute and chronic GVHD. Compared to Fli-1KO or Fli-1WT T cells, Fli-1HET T cells induced the mildest aGVHD and cGVHD supported by lowest Th1 and Th17 differentiation (Fig. 1 A-B). In addition, Fli-1HET T cells were able to maintain a strong graft-versus-leukemia (GVL) effect while inducing significantly attenuated GVHD. Fli-1-deficient CD8+ T cells expressed comparable levels of cytolytic molecules, reduced levels of exhaustion markers, and differentiated more into memory precursor effector cells compared to WT CD8 T cells after been transferred into allogeneic recipients. To further understand how Fli-1 regulates T-cell gene transcription and heterogeneity, we performed scRNA seq analysis of donor T cells, including Fli-1WT, Fli-1HET and Fli-1KO, isolated from recipient spleens. The scRNA seq analysis on the donor T cells indicated a major difference in the transcriptional regulation by Fli-1 in CD4 versus CD8 T cells and Fli-1 gene dose dependent modification in transcriptional pathways in Fli-1Het vs Fli-1KOT cells. Fli-1 deemed to play a distinct role in regulating gene transcription in CD4 versus CD8 T cells, in that more activation and less exhaustion gene pathways were enriched in CD8 Fli-1 deficient cells, while fewer genes in Th1/Th17 pathogenic pathways were enriched in CD4 Fli-1 deficient cells (Fig. 1 C-D). Overall, Fli-1 deficiency increased gene enrichment in OXPHOS pathway in both CD4 and CD8 T cells, and reduced gene enrichment in glycolysis pathway majorly in CD4 T cells (Fig. 1 E-F). Consistent with the lowest pathogenicity of Fli-1Het T cells for GVHD induction, Fli-1Het CD4 T cells had the lowest Th1/Th17 pathway gene enrichment and TCR pathway downstream gene expression. Camptothecin, topotecan or etoposide exhibited action as potent Fli-1 inhibitors at low-doses. For translational purpose, we tested these drugs and found that each of these drugs significantly attenuated GVHD severity while preserving the GVL effect. The efficacy of camptothecin or etoposide at low doses was dependent of Fli-1 expression on donor T cells. Mechanistically, blocking Fli-1 suppressed activated effector T cells while sparing Tregs and had no side effect on hematopoietic stem cells. Importantly, using the same treatment schedule as post-transplantation cyclophosphamide (PTCy), two doses of camptothecin or etoposide administered on day 3 and 4 were sufficient for the prevention of GVHD and leukemia relapse, in contrast to GVHD mortality in some of those treated with bendamustine and leukemia relapse in some of those treated with PTCy or cyclosporine (Fig. 1 G-I). In addition, the beneficial effect of camptothecin was extended in GVHD induced by human T cells in a xenograft model. In conclusion, we provide the first evidence that Fli-1 plays a crucial role in alloreactive CD4 T-cell activation and differentiation, and that targeting Fli-1 may be an attractive strategy for treating GVHD without compromising GVL effect. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Xenopus Explants and Transplants.

There is a long tradition of testing the developmental potential and competency of different regions of the embryo by transplanting tissue to novel locations or by growing isolated tissue in explant culture. The protocols introduced here describe several types of embryological manipulations in Xenopus that are particularly useful for analyzing tissue inductive signals, cell migration, and organogenesis. These techniques draw upon classical approaches that are quite easy to perform in both Xenopus laevis and Xenopus tropicalis embryos.

An Oil-in-Water adjuvant significantly increased influenza A/H7N9 split virus Vaccine-Induced circulating follicular helper T (cTFH) cells and antibody responses

BackgroundUnadjuvanted A/H7N9 vaccines are poorly immunogenic. The immune response is improved with the addition of MF59, an oil-in-water adjuvant. However, the cellular immunologic responses of MF59-adjuvanted A/H7N9 vaccine are not fully understood. Methods37 participants were vaccinated with 2 doses of 2013 influenza A/H7N9 vaccine (at Days 1 and 21) with or without MF59 and enrolled in an immunology substudy. Responses were assessed at multiple timepoints (Days 0, 8, 21, 29, and 42) for hemagglutination inhibition (HAI) and neutralizing antibody (Neut) assays, memory B cell responses by enzyme-linked ImmunoSpot; circulating follicular helper T cells (cTFH) and CD4 + T cells by intracellular cytokine staining. ResultsMF59-adjuvanted influenza A/H7N9 vaccine induced significantly higher hemagglutination inhibition (HAI) and neutralizing antibody (Neut) responses when compared to unadjuvanted vaccine. The adjuvanted vaccine elicited significantly higher levels of Inducible T-cell Co-Stimulator (ICOS) expression by CXCR3+CXCR5+CD4+ cTFH cells, compared to unadjuvanted vaccine. The magnitude of increase in cTFH cells (from baseline to Day 8) and in IL-21 expressing CD154+CD4+ T cells (from baseline to Days 8 and 21) correlated with HAI (at Day 29) and Neut antibody (at Days 8 and 29) titers. The increase in frequency of IL-21 expressing CD154+CD4+T cells (from baseline to Day 21) correlated with memory B cell frequency (at Day 42). ConclusioncTFH activation is associated with HAI and Neut responses in recipients of MF59-adjuvanted influenza A/H7N9 vaccine relative to unadjuvanted vaccine. Future studies should focus on optimizing the cTFH response and use cTFH as an early biomarker of serological response to vaccination.This trial was registered at clinicaltrials.gov, trial number NCT01938742.

Immuno-PET Monitoring of CD8+ T Cell Infiltration Post ICOS Agonist Antibody Treatment Alone and in Combination with PD-1 Blocking Antibody Using a 89Zr Anti-CD8+ Mouse Minibody in EMT6 Syngeneic Tumor Mouse

PurposeThe presence and functional competence of intratumoral CD8+ T cells is often a barometer for successful immunotherapeutic responses in cancer. Despite this understanding and the extensive number of clinical-stage immunotherapies focused on potentiation (co-stimulation) or rescue (checkpoint blockade) of CD8+ T cell antitumor activity, dynamic biomarker strategies are often lacking. To help fill this gap, immuno-PET nuclear imaging has emerged as a powerful tool for in vivo molecular imaging of antibody targeting. Here, we took advantage of immuno-PET imaging using 89Zr-IAB42M1-14, anti-mouse CD8 minibody, to characterize CD8+ T-cell tumor infiltration dynamics following ICOS (inducible T-cell co-stimulator) agonist antibody treatment alone and in combination with PD-1 blocking antibody in a model of mammary carcinoma.Procedures.Female BALB/c mice with established EMT6 tumors received 10 µg, IP of either IgG control antibodies, ICOS agonist monotherapy, or ICOS/PD-1 combination therapy on days 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 and 48 h post IV dose of 89Zr IAB42M1-14. In addition to 89Zr-IAB42M1-14 uptake in tumor and tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images to identify treatment effects. Imaging mass cytometry (IMC) and immunohistochemistry (IHC) was performed at end of study.Results89Zr-IAB42M1-14 uptake in the tumor was observed by day 11 and was preceded by an increase in the TDLN as early as day 4. The spatial distribution of 89Zr-IAB42M1-14 was more uniform in the drug treated vs. control tumors, which had spatially distinct tracer uptake in the periphery relative to the core of the tumor. IMC analysis showed an increased percentage of cytotoxic T cells in the ICOS monotherapy and ICOS/PD-1 combination group compared to IgG controls. Additionally, temporal radiomics analysis demonstrated early predictiveness of imaging features.ConclusionTo our knowledge, this is the first detailed description of the use of a novel immune-PET imaging technique to assess the kinetics of CD8+ T-cell infiltration into tumor and lymphoid tissues following ICOS agonist and PD-1 blocking antibody therapy. By demonstrating the capacity for increased spatial and temporal resolution of CD8+ T-cell infiltration across tumors and lymphoid tissues, these observations underscore the widespread potential clinical utility of non-invasive PET imaging for T-cell-based immunotherapy in cancer.