Expression Of Alpha Chain Research Articles

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy.

Understanding the interplay between the HIV reservoir and the host immune system may yield insights into HIV persistence during antiretroviral therapy (ART) and inform strategies for a cure. Here, we applied machine learning (ML) approaches to cross-sectional high-parameter HIV reservoir and immunology data in order to characterize host-reservoir associations and generate new hypotheses about HIV reservoir biology. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and measurement of genetically intact and total HIV proviral DNA frequencies were performed on peripheral blood samples from 115 people with HIV (PWH) on long-term ART. Analysis demonstrated that both intact and total proviral DNA frequencies were positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A leave-one-covariate-out inference approach identified specific HIV reservoir and clinical-demographic parameters, such as age and biological sex, that were particularly important in predicting immunophenotypes. Overall, immune parameters were more strongly associated with total HIV proviral frequencies than intact proviral frequencies. Uniquely, however, expression of the IL-7 receptor alpha chain (CD127) on CD4 T cells was more strongly correlated with the intact reservoir. Unsupervised dimension reduction analysis identified two main clusters of PWH with distinct immune and reservoir characteristics. Using reservoir correlates identified in these initial analyses, decision tree methods were employed to visualize relationships among multiple immune and clinical-demographic parameters and the HIV reservoir. Finally, using random splits of our data as training-test sets, ML algorithms predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA . The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology.

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy

Understanding the interplay between the HIV reservoir and the host immune system may yield insights into HIV persistence during antiretroviral therapy (ART) and inform strategies for a cure. Here, we applied machine learning (ML) approaches to cross-sectional high-parameter HIV reservoir and immunology data in order to characterize host–reservoir associations and generate new hypotheses about HIV reservoir biology. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and measurement of genetically intact and total HIV proviral DNA frequencies were performed on peripheral blood samples from 115 people with HIV (PWH) on long-term ART. Analysis demonstrated that both intact and total proviral DNA frequencies were positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A leave-one-covariate-out inference approach identified specific HIV reservoir and clinical–demographic parameters, such as age and biological sex, that were particularly important in predicting immunophenotypes. Overall, immune parameters were more strongly associated with total HIV proviral frequencies than intact proviral frequencies. Uniquely, however, expression of the IL-7 receptor alpha chain (CD127) on CD4 T cells was more strongly correlated with the intact reservoir. Unsupervised dimension reduction analysis identified two main clusters of PWH with distinct immune and reservoir characteristics. Using reservoir correlates identified in these initial analyses, decision tree methods were employed to visualize relationships among multiple immune and clinical–demographic parameters and the HIV reservoir. Finally, using random splits of our data as training-test sets, ML algorithms predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA . The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology.

Altered IL-7 signaling in CD4+ T cells from patients with visceral leishmaniasis.

CD4+ T cells play a central role in control of L. donovani infection, through IFN-γ production required for activation of macrophages and killing of intracellular parasites. Impaired control of parasites can in part be explained by hampered CD4+ T cells effector functions in visceral leishmaniasis (VL) patients. In a recent studies that defined transcriptional signatures for CD4+ T cells from active VL patients, we found that expression of the IL-7 receptor alpha chain (IL-7Rα; CD127) was downregulated, compared to CD4+ T cells from endemic controls (ECs). Since IL-7 signaling is critical for the survival and homeostatic maintenance of CD4+ T cells, we investigated this signaling pathway in VL patients, relative to ECs. CD4+ T cells were enriched from peripheral blood collected from VL patients and EC subjects and expression of IL7 and IL7RA mRNA was measured by real time qPCR. IL-7 signaling potential and surface expression of CD127 and CD132 on CD4+ T cell was analyzed by multicolor flow cytometry. Plasma levels of soluble IL-7 and sIL-7Rα were measured by ELISA. Transcriptional profiling data sets generated previously from our group showed lower IL7RA mRNA expression in VL CD4+ T cells as compared to EC. A significant reduction was, however not seen when assessing IL7RA mRNA by RT-qPCR. Yet, the levels of soluble IL-7Rα (sIL-7Rα) were reduced in plasma of VL patients compared to ECs. Furthermore, the levels of soluble IL-7 were higher in plasma from VL patients compared to ECs. Interestingly, expression of the IL-7Rα protein was higher on VL patient CD4+ T cells as compared to EC, with activated CD38+ CD4+ T cells showing higher surface expression of IL-7Rα compared to CD38- CD4+ T cells in VL patients. CD4+ T cells from VL patients had higher signaling potential baseline and after stimulation with recombinant human IL-7 (rhIL-7) compared to EC, as measured by phosphorylation of STAT5 (pSTAT5). Interestingly, it was the CD38 negative cells that had the highest level of pSTAT5 in VL patient CD4+ T cells after IL-7 stimulation. Thus, despite unaltered or potentially lowered IL7RA mRNA expression by CD4+ T cells from VL patients, the surface expression of the IL-7Rα was higher compared to EC and increased pSTAT5 was seen following exposure to rhIL-7. Accordingly, IL-7 signaling appears to be functional and even enhanced in VL CD4+ T cells and cannot explain the impaired effector function of VL CD4+ T cells. The enhanced plasma IL-7 may serve as part of homeostatic feedback mechanism regulating IL7RA expression in CD4+ T cells.

Vitamin D3 improved hypoxia-induced lung injury by inhibiting the complement and coagulation cascade and autophagy pathway

BackgroundPulmonary metabolic dysfunction can cause lung tissue injury. There is still no ideal drug to protect against hypoxia-induced lung injury, therefore, the development of new drugs to prevent and treat hypoxia-induced lung injury is urgently needed. We aimed to explore the ameliorative effects and molecular mechanisms of vitamin D3 (VD3) on hypoxia-induced lung tissue injury.MethodsSprague–Dawley (SD) rats were randomly divided into three groups: normoxia, hypoxia, and hypoxia + VD3. The rat model of hypoxia was established by placing the rats in a hypobaric chamber. The degree of lung injury was determined using hematoxylin and eosin (H&E) staining, lung water content, and lung permeability index. Transcriptome data were subjected to differential gene expression and pathway analyses. In vitro, type II alveolar epithelial cells were co-cultured with hepatocytes and then exposed to hypoxic conditions for 24 h. For VD3 treatment, the cells were treated with low and high concentrations of VD3.ResultsTranscriptome and KEGG analyses revealed that VD3 affects the complement and coagulation cascade pathways in hypoxia-induced rats, and the genes enriched in this pathway were Fgb/Fga/LOC100910418. Hypoxia can cause increases in lung edema, inflammation, and lung permeability disruption, which are attenuated by VD3 treatment. VD3 weakened the complement and coagulation cascade in the lung and liver of hypoxia-induced rats, characterized by lower expression of fibrinogen alpha chain (Fga), fibrinogen beta chain (Fgb), protease-activated receptor 1 (PAR1), protease-activated receptor 3 (PAR3), protease-activated receptor 4 (PAR4), complement (C) 3, C3a, and C5. In addition, VD3 improved hypoxic-induced type II alveolar epithelial cell damage and inflammation by inhibiting the complement and coagulation cascades. Furthermore, VD3 inhibited hypoxia-induced autophagy in vivo and in vitro, which was abolished by the mitophagy inducer, carbonyl cyanide-m-chlorophenylhydrazone (CCCP).ConclusionVD3 alleviated hypoxia-induced pulmonary edema by inhibiting the complement and coagulation cascades and autophagy pathways.

Cardiac complications in thalassemia throughout the lifespan: Victories and challenges.

Thalassemias are among the most common hereditary diseases in the world because heterozygosity offers protection against malarial infection. Affected individuals have variable expression of alpha or beta chains that lead to their unbalanced utilization during hemoglobin formation, oxidative stress, and apoptosis of red cell precursors prior to maturation. Some individuals produce sufficient hemoglobin to survive but suffer the vascular stress imposed by chronic anemia and ineffective erythropoiesis. In other patients, mature red cell formation is insufficient, and chronic transfusions are required-suppressing anemia and ineffective erythropoiesis but at the expense of iron overload. The cardiovascular consequences of thalassemia have changed dramatically over the previous five decades because of evolving treatment practices. This review summarizes this evolution, focusing on complications and management pertinent to modern patient cohorts.

Interleukin-6 trans-signaling induced laminin switch contributes to reduced trans-endothelial migration of granulocytic cells

Background and aimsLaminins are essential components of the endothelial basement membrane, which predominantly contains LN421 and LN521 isoforms. Regulation of laminin expression under pathophysiological conditions is largely unknown. In this study, we aimed to investigate the role of IL-6 in regulating endothelial laminin profile and characterize the impact of altered laminin composition on the phenotype, inflammatory response, and function of endothelial cells (ECs). MethodsHUVECs and HAECs were used for in vitro experiments. Trans-well migration experiments were performed using leukocytes isolated from peripheral blood of healthy donors. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analyzed using Microarray/qPCR and proximity extension assay, ELISA, immunostaining or immunoblotting techniques, respectively. ResultsStimulation of ECs with IL-6+sIL-6R, but not IL-6 alone, reduces expression of laminin α4 (LAMA4) and increases laminin α5 (LAMA5) expression at the mRNA and protein levels. In addition, IL-6+sIL-6R stimulation of ECs differentially regulates the release of several proteins including CXCL8 and CXCL10, which collectively were predicted to inhibit granulocyte transmigration. Experimentally, we demonstrated that granulocyte migration is inhibited across ECs pre-treated with IL-6+sIL-6R. In addition, granulocyte migration across ECs cultured on LN521 was significantly lower compared to LN421. In human atherosclerotic plaques, expression of endothelial LAMA4 and LAMA5 is significantly lower compared to control vessels. Moreover, LAMA5-to-LAMA4 expression ratio was negatively correlated with granulocytic cell markers (CD177 and myeloperoxidase (MPO)) and positively correlated with T-lymphocyte marker CD3. ConclusionsWe showed that expression of endothelial laminin alpha chains is regulated by IL-6 trans-signaling and contributes to inhibition of trans-endothelial migration of granulocytic cells. Further, expression of laminin alpha chains is altered in human atherosclerotic plaques and is related to intra-plaque abundance of leukocyte subpopulations.

Preclinical Development of Non-Edited CD7 CAR-Modified Invariant NKT Cell Therapy for T-Cell Malignancies and Acute Myeloid Leukemia

The major hurdle in developing chimeric antigen receptor T (CAR-T) cell therapy for T-cell malignancies is CAR-T cell fratricide. This refers to the self-killing of the CAR-T cells due to shared expression of targetable antigens (e.g., CD7) between CAR-T and malignant target T cells. This CAR-T fratricide creates difficulties in producing sufficient numbers of CAR-T cells for infusion and can limit survival of the cells in vivo. Nuclease (e.g., CRISPR)-mediated genomic disruption of CD7 or T cell receptor alpha chain expression provides an alternative approach to enabling expansion of CD7 CAR transduced T cells from allogeneic healthy donors. However, the safety and clinical implications of CRISPR-based genome editing remain unclear. Thus, other approaches to development of allogeneic, non-edited CD7 CAR-T cell therapy for relapsed and refractory T-cell malignancies is warranted. We unexpectedly found that human invariant natural killer T (iNKT) cells in normal blood donors displayed on average approximately 56% CD7 positivity (range 38 - 74%, n = 9 donors). Therefore, we hypothesize that CD7- subsets in iNKT cells expressing CD7 CAR can partially avoid fratricide without nuclease-mediated genome editing to delete CD7 and be expanded for adoptive cell therapy of CD7+ malignancies. By leveraging our proprietary 6000-10000-fold iNKT cell expansion platform, lentiviral transduced CD7 CAR iNKT (CD7 CAR-iNKT) cells can be expanded to clinical scale (n=2) (Fig. 1A). Based on CD19 CAR- and CD7 CAR-iNKT expansion rate (n=5), 100-500 doses (assuming 1-3 infusions per patient) of CAR-iNKT cells can be manufactured from a single donor-derived leukocyte concentrate. CD7 CAR-iNKT cells (n=5, 64-98% CAR+) specifically killed luciferase-expressing CD7+ T-cell acute lymphoblastic leukemia (T-ALL) (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1) at E/T ratios ranging from 20:1 to 2.2:1 compared to mock iNKT cells and CD19 CAR-iNKT cells (p < 0.0001). Control CD19 CAR-iNKT cells (n=3) specifically killed CD19+ B-cell tumor cells but not CD19- T-ALL and AML cells (p < 0.0001). Mock iNKT cells showed minimal cytotoxicity to all target cells except for CD1d+ Jurkat. CD7 CAR-iNKT cells (n=5) also produced significant amounts of IFN-γ in response to CD7+ T-ALL (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1, Kasumi-6) compared to mock (n=5, p < 0.001) and CD19 CAR-iNKT cells (p < 0.05 except for Jurkat, p < 0.01). CD19 CAR-iNKT cells (n=3) produced significant amounts of IFN-γ to CD19+ B-cell leukemia and lymphoma cell lines (Nalm6, Daudi, Raji) compared to mock and CD7 CAR-iNKT cells (p < 0.0001). As expected, CD7 CAR-iNKT cells significantly eliminated almost all CD7+ cells (mean ± SD 0.4% ± 0.1, n=4) compared to mock (61.6% ± 24.4, n=4) and CD19 or ROR1 CAR-iNKT cells (69.9% ± 24.4, n=3) (p < 0.0001). Furthermore, expanded CD7 CAR-iNKT cells were >95% CD3+TCRVα24-Jα18+ and specifically responded to a-galactosylceramide or 7DW8-5 lipid antigens presented by CD1d molecules. The anti-leukemic effect of CD7 CAR-modified iNKT cells was assessed in HSB2 T-ALL and KG1 AML xenograft models (n=5 per group). Three i.v. infusions of thawed CD7 CAR-iNKT cells (5x106 cells/mouse) at days 0, 3, and 6 into leukemia (5x105/mouse, i.v. at day -2)-bearing mice significantly suppressed T-ALL growth by bioluminescent imaging analysis at days 3 (p < 0.0001), 6 (p=0.0001-0.0005), 10 (p=0.0012-0.0024) and 17 (p < 0.0001) compared to PBS and mock controls (Fig. 1B). A significant survival advantage was observed after CD7 CAR-iNKT cell therapy compared to PBS and mock controls (p = 0.0009, Fig. 1C). Two i.v. infusions of thawed CD7 CAR-iNKT cells into pre-established AML mice significantly suppressed AML growth at days 3 (p = 0.0096), 6 (p < 0.0001), 10 (p = 0.0026), 17 (p < 0.0001), 24 (p < 0.0001), 31 (p < 0.0001), 38 (p < 0.0001) and 45 (p = 0.0003) compared to mock control (Fig. 1D). CD7 CAR-iNKT cell therapy also significantly prolonged the animal survival (p = 0.0069, Fig. 1E). In conclusion, these results demonstrate that CD7 CAR-iNKT cells without genome editing can be manufactured to therapeutic doses and mount anti-CD7+ tumor responses in vitro and in vivo. Our CD7 CAR-iNKT cells can be used as allogeneic, off-the-shelf products to treat all subtypes of T-ALL and 30% of CD7+ AML patients. In addition, our CD7 CAR-iNKT cells are likely more effective in 70% CD7+CD1d+ T-ALL patients as they provide dual target specificity and reduce the likelihood of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FGA Controls VEGFA Secretion to Promote Angiogenesis by Activating the VEGFR2-FAK Signalling Pathway.

BackgroundOur previous work revealed the high expression of fibrinogen alpha chain (FGA) in patients with endometriosis (EM) and that it could promote the migration and invasion of endometrial stromal cells. Angiogenesis is the key condition for the development of EM. This study was aimed to elucidate the role of FGA in endometrial stromal cells involved in angiogenesis in EM.MethodsImmunohistochemistry was used to detect the microvessel density (MVD) and VEGF expression in the eutopic endometrium samples from EM and non-EM. The conditioned medium (CM) of human primary eutopic endometrial stromal cells (EuESC) and immortalized endometrial stromal cell line hEM15A with FGA knockdown were collected and used to treat human umbilical vein endothelial cells (HUVECs). Then, tube formation assay, EdU assay, wound assay, transwell assay and flow cytometry assays were performed to assess the function of HUEVCs in vitro. The angiogenic capability of HUVECs was further measured using a matrigel plug assay with BALB/c nude mice in vivo. Immunofluorescence was used to detect the expression of F-actin and VE-cadherin. RT-PCR and western blotting were used to detect the expression of angiogenesis-related factors in endometrial stromal cells and downstream signalling pathways in HUVECs.ResultsMVD and VEGF expression in the eutopic endometrium of EM patients were significantly higher than those in the normal endometrium of non-EM patients, and the increased MVD in EM indicates an increased risk of recurrence. Functionally, we found that CM of endometrial stromal cells with FGA knockdown could inhibit HUEVCs migration and tube formation in vitro and in vivo, while having no significant effect on HUVECs proliferation, apoptosis and cell cycle. Mechanically, the expression of VEGFA, PDGF, FGF-B, VEGF, MMP-2 and MMP-9 was reduced in hEM15A cells with FGA knockdown. CM of hEM15A cells with FGA knockdown reduced the number of microfilaments and pseudopodia, as well as the expression of VE-cadherin, and inhibited the activity of VEGFR2 and the FAK signalling pathway in HUVECs.ConclusionOur study demonstrated FGA could enhance the interaction between endometrial stromal cells and HUVECs via the potential VEGA-VEGFR-FAK signalling axis and promote EM angiogenesis, revealing a promising therapeutic approach for EM.

Heritable changes in division speed accompany the diversification of single T cell fate

Rapid clonal expansion of antigen-specific T cells is a fundamental feature of adaptive immune responses. It enables the outgrowth of an individual T cell into thousands of clonal descendants that diversify into short-lived effectors and long-lived memory cells. Clonal expansion is thought to be programmed upon priming of a single naive T cell and then executed by homogenously fast divisions of all of its descendants. However, the actual speed of cell divisions in such an emerging "T cell family" has never been measured with single-cell resolution. Here, we utilize continuous live-cell imaging invitro to track the division speed and genealogical connections of all descendants derived from a single naive CD8+ T cell throughout up to ten divisions of activation-induced proliferation. This comprehensive mapping of T cell family trees identifies a short burst phase, in which division speed is homogenously fast and maintained independent of external cytokine availability or continued T cell receptor stimulation. Thereafter, however, division speed diversifies, and model-based computational analysis using a Bayesian inference framework for tree-structured data reveals a segregation into heritably fast- and slow-dividing branches. This diversification of division speed is preceded already during the burst phase by variable expression of the interleukin-2 receptor alpha chain. Later it is accompanied by selective expression of memory marker CD62L in slower dividing branches. Taken together, these data demonstrate that T cell clonal expansion is structured into subsequent burst and diversification phases, the latter of which coincides with specification of memory versus effector fate.

Critical COVID-19 is associated with distinct leukocyte phenotypes and transcriptome patterns.

BackgroundPrognostic markers for disease severity and identification of therapeutic targets in COVID‐19 are urgently needed. We have studied innate and adaptive immunity on protein and transcriptomic level in COVID‐19 patients with different disease severity at admission and longitudinally during hospitalization.MethodsPeripheral blood mononuclear cells (PBMCs) were collected at three time points from 31 patients included in the Norwegian SARS‐CoV‐2 cohort study and analysed by flow cytometry and RNA sequencing. Patients were grouped as either mild/moderate (n = 14), severe (n = 11) or critical (n = 6) disease in accordance with WHO guidelines and compared with patients with SARS‐CoV‐2‐negative bacterial sepsis (n = 5) and healthy controls (n = 10).ResultsCOVID‐19 severity was characterized by decreased interleukin 7 receptor alpha chain (CD127) expression in naïve CD4 and CD8 T cells. Activation (CD25 and HLA‐DR) and exhaustion (PD‐1) markers on T cells were increased compared with controls, but comparable between COVID‐19 severity groups. Non‐classical monocytes and monocytic HLA‐DR expression decreased whereas monocytic PD‐L1 and CD142 expression increased with COVID‐19 severity. RNA sequencing exhibited increased plasma B‐cell activity in critical COVID‐19 and yet predominantly reduced transcripts related to immune response pathways compared with milder disease.ConclusionCritical COVID‐19 seems to be characterized by an immune profile of activated and exhausted T cells and monocytes. This immune phenotype may influence the capacity to mount an efficient T‐cell immune response. Plasma B‐cell activity and calprotectin were higher in critical COVID‐19 while most transcripts related to immune functions were reduced, in particular affecting B cells. The potential of these cells as therapeutic targets in COVID‐19 should be further explored.

High Expression of Interleukin-3 Receptor Alpha Chain (CD123) Predicts Favorable Outcome in Pediatric B-Cell Acute Lymphoblastic Leukemia Lacking Prognosis-Defining Genomic Aberrations.

BackgroundAberrant expression of CD123 (IL-3Rα) was observed in various hematological malignancies including acute lymphoblastic leukemia (ALL), which is the most common malignancy in childhood. Although widely used for minimal residual disease (MRD) monitoring, the prognostic value of CD123 has not been fully characterized in pediatric B-ALL. This retrospective study aims to evaluate the association between the CD123 expression of leukemic blasts and the outcomes of the pediatric B-ALL patients.MethodsA total of 976 pediatric B-ALL, including 328 treated with CCLG-ALL-2008 protocol and 648 treated with CCCG-ALL-2015 protocol, were recruited in this retrospective study. CD123 expression was evaluated by flow cytometry. Patients with >50, 20–50, or <20% of CD123 expressing blasts were grouped into CD123high, CD123low, and CD123neg, respectively. The correlation between CD123 expression and the patients’ clinical characteristics, overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were studied statistically.ResultsOf 976 pediatric B-ALL, 53.4% from the CCLG-ALL-2008 cohort and 49.2% from the CCCG-ALL-2015 cohort were CD123high. In the CCLG-ALL-2008 cohort, CD123high was significantly associated with chromosome hyperdiploidy (p < 0.0001), risk stratification (p = 0.004), and high survival rate (p = 0.005). By comparing clinical outcomes, patients with CD123high displayed favorable prognosis, with a significantly better OS (p = 0.005), EFS (p = 0.017), and RFS (p = 0.045), as compared to patients with CD123low and CD123neg. The prognostic value of CD123 expression was subsequently confirmed in the CCCG-ALL-2015 cohort. Univariate and multivariate cox regression model analysis showed that high CD123 expression was independently associated with favorable EFS (OR: 0.528; 95% CI: 0.327 to 0.853; p = 0.009) in this cohort. In patients without prognosis-defining genomic abnormalities, high CD123 expression strongly indicated superior survival rates and was identified as an independent prognosis factor for EFS and RFS in both cohorts.ConclusionsA group of B-ALL lacks prognosis-defining genomic aberrations, which proposes a challenge in risk stratification. Our findings revealed that high CD123 expression of leukemic blasts was associated with favorable clinical outcomes in pediatric B-ALL and CD123 could serve as a promising prognosis predictor, especially in patients without prognosis-defining genetic aberrations.

A Bioassay-Based Approach for the Batch-To-Batch Consistency Evaluation of Xuesaitong Injection on a Zebrafish Thrombosis Model.

Quality control of Chinese medicine (CM) is mainly based on chemical testing, which sometimes shows weak correlation to pharmacological effects. Thus, there is a great demand to establish bioactivity-based assays to ensure the quality of CM. The aim of the present study was to establish a bioassay-based approach to evaluate the biological activity of Xuesaitong injection (XST) based on an in vivo zebrafish model. Zebrafish larvae with arachidonic acid (AA)-induced thrombus were applied to evaluate anti-thrombosis effects of XST and explore the potential mechanism of XST. Analysis of major components in normal and abnormal XST samples was performed by high performance liquid chromatography (HPLC). The results indicate that XST could significantly restore heart red blood cells (RBCs) intensity of thrombotic zebrafish in a dose-dependent manner, whilst decreasing RBCs accumulation in the caudal vein. The results were confirmed using a green fluorescence protein (GFP)-labeled zebrafish thrombosis model. Moreover, we could show that XST downregulates the expression of the fibrinogen alpha chain (fga) gene to inhibit the coagulation cascade during the process of thrombosis in zebrafish. Notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rd, which were considered to be the major components of XST, also showed moderate anti-thrombosis efficacy. Further results showed that the zebrafish thrombosis model could efficiently distinguish five abnormal batches of XST from 24 normal batches. Furthermore, the inhibition rates of different batches were correlated with the content level of major components. Our results suggested that the proposed zebrafish thrombosis model could be successfully used to evaluate the batch-to-batch consistency of XST, which provided an alternative way for the quality control of CM.

NK/T cell ratios associate with interleukin-2 receptor alpha chain expression and shedding in multiple sclerosis

NK/T-cell ratios predict disease activity in relapsing remitting multiple sclerosis (RRMS). We investigated in 50 RRMS patients whether interleukin-2 receptor alpha-chain (IL-2Rα) expression and shedding associates with NK/T-cell balance, as suggested by daclizumab-trials in RRMS. A subsample (N = 31) was genotyped for IL2RA-associated MS risk SNPs. CD56bright NK-cell/IL-17A+CD4+ T-cell ratios correlated negatively with plasma and PBMC-culture supernatant sIL-2Rα-levels [R = -0.209; p = 0.038 and R = -0.254; p = 0.012, resp.], and with CD4+ T-cell CD25 MFI [R = -0.341; p = 0.001]. Carriers of the rs3118470 risk-allele showed higher sIL-2Rα-levels (P = 0.031) and a lower CD56bright NK-cell/IL-17A+CD4+ T-cell ratio (P = 0.038). Therefore, IL-2Rα may be involved in the interplay between NK-cells and T-cells.

Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins.

Among all T and NK cell subsets, regulatory T (Treg) cells typically respond to the lowest concentrations of IL-2 due to elevated surface expression of the IL-2R alpha chain (IL2RA; CD25) and the high affinity IL-2 receptor (IL-2R) complex. This enhanced sensitivity forms the basis for low-dose (LD) IL-2 therapy for the treatment of inflammatory diseases, where efficacy correlates with increased Treg cell number and expression of functional markers. Despite strong preclinical support for this approach, moderate and variable clinical efficacy has raised concerns that adequate Treg selectivity still cannot be achieved with LD IL-2, and/or that doses are too low to stimulate effective Treg-mediated suppression within tissues. This has prompted development of IL-2 variants with greater Treg selectivity, achieved through attenuated affinity for the signaling chains of the IL-2R complex (IL2RB or CD122 and IL2RG or CD132) and, consequently, greater reliance on high CD25 levels for full receptor binding and signaling. While certain IL-2 variants have advanced to the clinic, it remains unknown if the full range of IL-2R signaling potency and Treg-selectivity observed with low concentrations of wildtype IL-2 can be sufficiently recapitulated with attenuated IL-2 muteins at high concentrations. Using a panel of engineered IL-2 muteins, we investigated how a range of IL-2R signaling intensity, benchmarked by the degree of STAT5 phosphorylation, relates to biologically relevant Treg cell responses such as proliferation, lineage and phenotypic marker expression, and suppressor function. Our results demonstrate that a surprisingly wide dynamic range of IL-2R signaling intensity leads to productive biological responses in Treg cells, with negligible STAT5 phosphorylation associating with nearly complete downstream effects such as Treg proliferation and suppressor activity. Furthermore, we show with both in vitro and humanized mouse in vivo systems that different biological responses in Treg cells require different minimal IL-2R signaling thresholds. Our findings suggest that more than minimal IL-2R signaling, beyond that capable of driving Treg cell proliferation, may be required to fully enhance Treg cell stability and suppressor function in vivo.

Super2-expressing CAR T cells generate improved anti-tumor responses against solid tumors

Abstract Chimeric antigen receptors (CAR) augment tumor specific immune responses by redirecting all T cells towards recognition of tumor antigen. Although CAR T cell treatment shows promising therapeutic effects in leukemia patients, current CAR therapies are inadequate against solid tumors, in part from exclusion or functional exhaustion in the suppressive microenvironment. Exogenously administered Interleukin 2 (IL-2) improves efficacy and expansion of CAR T cell therapy. However, IL-2 administration is limited by high toxicity and low half-life. Super2 is an engineered IL-2 variant that binds with high affinity to the beta and gamma chains of the IL-2 receptor even without IL-2R alpha chain expression. We found that Super2 leads to increased phosphorylation of STAT-5 and more effectively activates lymphocyte populations compared to endogenous IL-2 in vitro. We hypothesized that engineering CAR T cells to express Super2 will improve their functionality within the tumor microenvironment. CAR T cell-intrinsic Super2 may also increase activation of the endogenous tumor-reactive T cells resulting in enhanced tumor control and the establishment of long-term memory even after loss of CAR T cells. To test this hypothesis, we designed CAR T cells that produce Super2 constitutively or in response to CAR stimulation. We found that Super2-expressing CAR T cells increased tumor infiltrating leukocytes (TILs) compared to CAR T cell treatment alone in immune competent murine models. Additionally, Super2-expressing CAR T cells delayed B16F10 melanoma growth in comparison to CAR T cell alone. We conclude that Super2-expressing CAR T cells provide improved protection against solid tumors.

Membrane-associated RING-CH (MARCH) proteins down-regulate cell surface expression of the interleukin-6 receptor alpha chain (IL6Rα).

Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses.

CD73 Rather Than CD39 Is Mainly Involved in Controlling Purinergic Signaling in Calcified Aortic Valve Disease.

The study aimed to compare composition of peripheral blood T-cell subsets and assess their surface expression of CD39 and CD73 ectonucleotidases in patients with severe and moderate aortic stenosis (AS) as well as to evaluate involvement of T-cell-mediated immune processes in valve calcification. The study was performed with 38 patients suffering from severe calcified aortic stenosis (SAS), 33 patients with MAS, and 30 apparently healthy volunteers (HVs). The relative distribution and percentage of T-cell subsets expressing CD39 and CD73 were evaluated by flow cytometry. T helper (Th) and cytotoxic T-cell subsets (Tcyt) were identified by using CD3, CD4, and CD8 antibodies. Regulatory T cells (Tregs) were characterized by the expression of CD3, CD4, and high IL-2R alpha chain (CD25high) levels. CD45R0 and CD62L were used to assess differentiation stage of Th, Tcyt, and Treg subsets. It was found that MAS and SAS patients differed in terms of relative distribution of Tcyt and absolute number of Treg. Moreover, the absolute number of Tcyt and terminally differentiated CD45RA-positive effector T-cells (TEMRA) subset was significantly higher in SAS vs. MAS patients and HVs. However, the absolute and relative number of naïve Th and the absolute number of Treg were significantly higher in MAS vs. SAS patients; the relative number of naïve Tregs was significantly (p < 0.01) decreased in SAS patients. It was shown that CD73 expression was significantly higher in SAS vs. MAS patients noted in all EM, CM, TEMRA, and naïve Th cell subsets. However, only the latter were significantly increased (p = 0.003) in patients compared with HVs. SAS vs. MAS patients were noted to have significantly higher percentage of CD73+ EM Tcyt (p = 0.006) and CD73+ CM Tcyt (p = 0.002). The expression of CD73 in patients significantly differed in all three Treg populations such as EM (p = 0.049), CM (p = 0.044), and naïve (p < 0.001). No significant differences in CD39 expression level was found in MAS and SAS patients compared with the HV group. Overall, the data obtained demonstrated that purinergic signaling was involved in the pathogenesis of aortic stenosis and calcification potentially acting via various cell types, wherein among enzymes, degrading extracellular ATP CD73 rather than CD39 played a prominent role.

The VLA-4 Integrin Is Constitutively Activated in a Subset of CD49d-Expressing CLL: A Relationship with the Autonomous BCR-Mediated Signaling?

Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of tumor cell-microenvironment interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its specific ligands. In this context, VLA-4 needs to be maintained in an activated state by a continuous stream of inside-out signals from the BCR, which can be triggered by canonical antigens as well as in an autonomous antigen-independent manner, a BCR-mediated signaling recently emphasized to specifically occur in CLL cells.Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this still not described feature with the presence of signals from the BCR continuously activating VLA-4.Methods. Expression of the integrin alpha (CD49c, CD49d, CD49e) and beta1 (CD29) chains was analyzed by flow cytometry. The VLA-4 activation state was investigated by flow cytometry using the conformational sensitive anti-CD29 monoclonal antibody HUTS21, employed in conjunction with sources of VLA-4 ligands: either autologous plasma-containing fibronectin (FN) and soluble (s) VCAM-1 or increasing concentrations of exogenously added LDV-containing peptides as a VLA-4 specific ligand. In detail: i) HUTS21 expression was analyzed using whole blood (WB) samples from 727 CLL patients. Negative controls were obtained after plasma depletion through washing of WB samples; ii) the VLA-4 activation state expressed as receptor occupancy (RO) (J Biol Chem, 284,14337, 2009) was analyzed in sequential (t=0, day14, day 30) frozen/thawed samples from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=16) and from a clinical trial (NCT02827617, n=14). BCR engagement was performed using goat F(ab′)2 anti-human IgM. ELISA assays were used to quantify FN and sVCAM-1 in plasma samples.Results. According to the 30% cutoff, CD49d was expressed in 351/727 (48%), while CD29 was expressed in 676/727 (93%), and a strong correlation between CD49d and CD29 was observed (rho=0.7, p<0.0001). Among CD49d positive CLL cases, 81/351 (23%) expressed a constitutively activated form of VLA-4 identified by >20% (arbitrary cut-off) HUTS21 expression. Notably, HUTS21 positive expression was not circumscribed to VLA-4 expressing CLL, being also detected in 64/375 (17%) CD49d-, CD49c+ and/or CD49e+ CLL, thus envisioning a role of other integrins (CD49c and CD49e) in CLL cell interactions in tissues sites. HUTS21 expression did not correlate with a definite cytogenetic group, IGHV mutational status or with a specific IGHV family and stereotype. Depletion of the plasma component from the WB samples before HUTS21 staining significantly reduced the proportion of HUTS21 positive cells compared with those measured in WB samples (p=0.001, n=11), suggesting the need of plasma-borne ligands for HUTS21 epitope exposure. Consistently, both FN (mean 350 μg/ml) and sVCAM-1 (mean 4.8 μg/ml) were detected in the plasma of CLL cases, irrespective to HUTS21 positivity. According to these observations, variable constitutive VLA-4 activation states were observed in CLL cells collected at the pre-treatment stage from patients undergoing IB treatment (VLA-4 RO ranging from 0.22 to 0.40); these values significantly decreased in CLL cells collected at day 14 (p=0.03) and day 30 (p=0.02) on IB, suggesting an IB-dependent impairment of antigen-independent (autonomous) BCR signals. The variability of the constitutive VLA-4 activation levels observed at pre-treatment was paralleled by variable VLA-4 activation levels upon BCR triggering (VLA-4 RO ranging from 0.56 to 0.65). Of note, an inverse correlation between the VLA-4 constitutive level and the extent of anti-IgM induced-VLA-4 activation was found (n=19, rho=-0.5, p=0.0093), implying a competition between antigen-independent and antigen-driven BCR signalling.Conclusion. The presence of a constitutively activated form of VLA-4 is observed in a subset of CLL which might be connected to continuous VLA-4 inside-out stimulation derived from an autonomous BCR signaling, which is currently under evaluation. DisclosuresZaja:Takeda: Honoraria; Sandoz: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria.

A Next-Generation Fc-Bearing CD3-Engaging Bispecific DART® Platform with Extended Pharmacokinetic and Expanded Pharmacologic Window: Characterization As CD123 x CD3 and CD19 x CD3 DART Molecules

Background: CD3-engaging DART molecules direct T cells to tumor-expressed antigens. Flotetuzumab, a CD123 x CD3 bispecific DART molecule that targets the differential expression of the IL-3 receptor alpha chain (CD123) on acute myeloid leukemia (AML) blasts and leukemic stem cells, is currently being investigated in a phase 1 study in relapsed/refractory AML and myelodysplastic syndrome (MDS), with initial evidence of clinical activity1. A common adverse event in redirected T-cell killing interventions is cytokine release syndrome (CRS), a Cmax-associated event that may limit their therapeutic window. Owing to its short circulating half-life, flotetuzumab is administered as continuous infusion, which affords prolonged exposure with limited Cmax excursions. In contrast, long-acting Fc-bearing, CD3-engaging molecules would eliminate the need for continuous dosing, but would be especially impacted by CRS, given the high Cmax levels required to maintain adequate trough concentrations over several days or weeks. We previously reported the development of a CD3-engaging DART molecule with reduced affinity for CD3 that maintained maximal target cell killing and T-cell proliferation, albeit at higher concentrations than its wild-type (WT) counterpart, but with reduced cytokine release2. We report here further optimization and validation of the approach.Methods: Anti-CD3 variants spanning a range of affinities were formatted as Fc-bearing DART molecules with CD123- or CD19-targeting arms and evaluated for cell binding as well as for antigen-dependent induction of T-cell proliferation, cytolytic activity and cytokine release. A therapeutic index (TI) was determined from the ratio of in vitro cell killing activity to that of target cell-induced cytokine release. In vivo anti-tumor activity was assessed in human immune cell-reconstituted mouse tumor models or in mice expressing the epitope recognized by the CD3 arm of the DART proteins (human CD3 k/i mice). Safety, pharmacokinetic (PK) and biological activity of selected molecules was evaluated in cynomolgus monkeys.Results: Six Fc-bearing DART molecules (from a panel of 23 CD123 x CD3 DART variants) were selected for characterization. These molecules spanned a relatively small interval of low CD3 affinities, but varied greatly in activity, from inactive at all concentrations tested to being capable of lysing tumor cells, including primary leukemic and MDS blasts, to the same maximal level of killing observed with the parental WT DART molecule. Higher concentrations of the active variants were needed, owing to a deliberate design incorporating decreased CD3 binding. At levels capable of complete tumor cell lysis, active DART variants showed greatly reduced pro-inflammatory cytokine release (e.g., IL-2, IFN-γ and TNF-α) in vitro and in human PBMC-reconstituted mice compared to the WT version. Molecules were ranked based on their TI and a version with a highly favorable index was engineered as a B-cell targeting CD19 x CD3 DART molecule to conveniently ascertain its biological activity in cynomolgus monkeys. The CD19 x CD3 DART variant demonstrated good tolerability at single doses up to 30 mg/kg. B-cell depletion in the circulation and lymphoid tissues occurred at 1 mg/kg and achieved maximal depletion at ≥10 mg/kg, equivalent to the degree of reduction observed with 0.1 mg/kg of the WT molecule. However, only minimal circulating pro-inflammatory cytokines (including IL-6) were observed at all doses of the DART variant, while a substantial increase in IL-2, IL-6 and IFN-γ (among others) was observed with 0.1 mg/kg of the WT counterpart. The PK profile of the DART variant in cynomolgus monkeys appeared linear in the 1-30 mg/kg interval, with a half-life of 5 to 10 days, consistent with possible clinical dosing at weekly or longer intervals.Conclusions: A next-generation CD3-engaging Fc-bearing DART platform was generated through CD3 arm affinity modulation. These DART variants feature extended PK and an expanded TI suitable for potential clinical development in hematological malignancies.1. Uy et al, Blood 2017, vol. 130, Suppl 1 6372. Huang et al, Keystone Symposia: Antibodies as Drugs, 2/25-3/1/2018, Whistler, BC, Canada DisclosuresBonvini:MacroGenics: Employment, Equity Ownership. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Huang:MacroGenics, Inc.: Employment, Equity Ownership. Lam:MacroGenics, Inc.: Employment, Equity Ownership. Kaufman:MacroGenics, Inc.: Employment, Equity Ownership. Liu:MacroGenics, Inc.: Employment, Equity Ownership. Alderson:MacroGenics, Inc.: Employment, Equity Ownership. Stahl:MacroGenics, Inc.: Employment, Equity Ownership. Brown:MacroGenics, Inc.: Employment, Equity Ownership. Li:MacroGenics, Inc.: Employment, Equity Ownership. Sharma:MacroGenics, Inc.: Employment, Equity Ownership. Tamura:MacroGenics, Inc.: Employment, Equity Ownership. Johnson:MacroGenics, Inc.: Consultancy, Equity Ownership. Moore:MacroGenics, Inc.: Employment, Equity Ownership.

IMMU-20. SINGLE CELL CYTOMICS OF PERIPHERAL BLOOD MONONUCLEAR CELLS REVEALS NEW AVENUES FOR GLIOMA IMMUNOTHERAPY

Glioblastoma is the most common primary brain tumor with a dismal prognosis and a lack of effective treatment options after relapse. Despite striking successes that were achieved in the last decade in other types of cancer, evidence of sustainable responses to T cell directed immunotherapy in glioblastoma remains limited. This might be explained by an emerging hypothesis that glioblastomas are cold tumours with poor lymphocyte infiltration. Therefore, some recent efforts have been focused on increasing cytotoxic lymphocyte infiltration. Here we present our workflow for subpopulation frequency, marker expression, and correlation network (INFERENCE) analysis, in this study applied to screen for possible new targets to enhance anti-glioblastoma immune responses. Using high-dimensional, single-cell mass cytometry (CyTOF) we profiled peripheral blood mononuclear cells derived from 160 patients with glioblastoma, lower grade glioma, or metastasis, meningioma and epilepsy as controls. Based on a CyTOF panel composed mainly of lymphocyte markers and check-point inhibition markers we could effectively dissect blood leukocytes into 100 phenotypically separate subsets of CD4 T cells (35 subsets), CD8 T cells (17 subsets), monocytes (12 subsets), NK cells (14 subsets), and B cells (15 subsets), and map their frequencies, phenotypes and correlation statistics. We thereby created an in depth human atlas of peripheral blood mononuclear cells in patients with different types of intracranial tumors. Interestingly, a subset of B cells characterized by high expression of the Interleukin-2 receptor alpha chain (CD25, a typical marker of regulatory T cells) was significantly increased in glioblastoma patients. This immune suppressive B cell subset has previously been associated with enhancing the suppressive effect of regulatory T cells. Further research is warranted to investigate the role of this aberrant B cell subset in the context of glioblastoma and its potential as target for immunotherapy.