ALBERT

Description: Genetic variation in the serotonin transporter gene (SLC6A4) is associated with vulnerability to affective disorders and pharmacotherapy efficacy. We recently identified sequence polymorphisms in the common marmoset SLC6A4 repeat region (AC/C/G and CT/T/C) associated with individual differences in anxiety-like trait, gene expression, and response to antidepressants. The mechanisms underlying the effects of these polymorphisms are unknown, but a key mediator of serotonin action is the serotonin 2A receptor (5HT2A). Thus, we correlated 5HT2A binding potential (BP) and RNA gene expression in 16 SLC6A4 genotyped marmosets with responsivity to 5HT2A antagonism during the human intruder test of anxiety. Voxel-based analysis and RNA measurements showed a reduction in 5HT2A BP and gene expression specifically in the right posterior insula of individuals homozygous for the anxiety-related variant AC/C/G. These same marmosets displayed an anxiogenic, dose-dependent response to the human intruder after 5HT2A pharmacological antagonism, while CT/T/C individuals showed no effect. A voxel-based correlation analysis, independent of SLC6A4 genotype, revealed that 5HT2A BP in the adjacent right anterior insula and insula proisocortex was negatively correlated with trait anxiety scores. Moreover, 5HT2A BP in both regions was a good predictor of the size and direction of the acute emotional response to the human intruder threat after 5HT2A antagonism. Our findings suggest that genetic variation in the SLC6A4 repeat region may contribute to the trait anxious phenotype via neurochemical changes in brain areas implicated in interoceptive and emotional processing, with a critical role for the right insula 5HT2A in the regulation of affective responses to threat.

Description: Sensors, Vol. 18, Pages 2583: Q3: A Compact Device for Quick, High Precision qPCR Sensors doi: 10.3390/s18082583 Authors: Marco Cereda Alessandro Cocci Davide Cucchi Lillo Raia Danilo Pirola Lorenzo Bruno Pietro Ferrari Valentina Pavanati Giorgia Calisti Francesco Ferrara Alessandro P. Bramanti Marco A. Bianchessi An accurate and easy-to-use Q3 system for on-chip quantitative real-time Polymerase Chain Reaction (qPCR) is hereby demonstrated, and described in detail. The qPCR reactions take place inside a single-use Lab-on-a-Chip with multiple wells, each with 5 to 15 µL capacity. The same chip hosts a printed metal heater coupled with a calibrated sensor, for rapid and accurate temperature control inside the reaction mixture. The rest of the system is non-disposable and encased in a 7 × 14 × 8.5 (height) cm plastic shell weighing 300 g. Included in the non-disposable part is a fluorescence read-out system featuring up to four channels and a self-contained control and data storage system, interfacing with an external user-friendly software suite. Hereby, we illustrate the engineering details of the Q3 system and benchmark it with seamlessly ported testing protocols, showing that Q3 equals the performance of standard commercial systems. Overall, to the best of our knowledge, this is one of the most mature general-purpose systems for on-chip qPCR currently available.

Description: A broad range hyper-spectroscopic microscope fed by a supercontinuum laser source and equipped with an almost achromatic optical layout is illustrated with detailed explanations of the design, implementation and data. The real novelty of this instrument, a confocal spectroscopic microscope capable of recording high resolution reflectance data in the VIS-IR spectral range from about 500 nm to 2.5 μm wavelengths, is the possibility of acquiring spectral data at every physical point as defined by lateral coordinates, X and Y, as well as at a depth coordinate, Z, as obtained by the confocal optical sectioning advantage. With this apparatus we collect each single scanning point as a whole spectrum by combining two linear spectral detector arrays, one CCD for the visible range, and one InGaAs infrared array, simultaneously available at the sensor output channel of the home made instrument. This microscope has been developed for biomedical analysis of human skin and other similar applications. Results are shown illustrating the technical performances of the instrument and the capability in extracting information about the composition and the structure of different parts or compartments in biological samples as well as in solid statematter. A complete spectroscopic fingerprinting of samples at microscopic level is shown possible by using statistical analysis on raw data or analytical reflectance models based on Abelés matrix transfer methods.

Description: Background: There are few therapeutic options for higher risk patients with myelodysplastic syndrome (MDS) who fail standard therapy, and their 2-year survival rate is approximately 15%. Here we report on a recently initiated collaborative (industry-academia) first-in-human phase 1 clinical trial to assess the safety and tolerability of a novel form of adoptive T cell immunotherapy for such patients that targets patient and disease-specific, mutation-derived neoantigens. This experimental therapy is based on the concept that a) cancer is caused by somatic mutations that may generate novel immunogenic proteins (ie, neopeptides and possible neoantigens), b) that the adaptive immune system can be trained ex vivo to recognize neopeptides as neoantigens and c) that infusion of culture-expanded, neoantigen-immunized autologous T cells may be safe and therapeutically effective. Methods: This is an open-label, phase 1, 3+3 dose escalation trial with 3 cell doses (0.3, 1, and 3.0 × 107nucleated cells/kg) in cohorts of 3 patients each (see www.clinicaltrials.gov, NCT-03258359). Eligible subjects are 18 years of age or older and will have Intermediate, High, or Very High risk MDS by the revised International Prognostic Scoring System, with at least one cytopenia, and will have failed or relapsed after 6 cycles of standard hypomethylating therapy or declined such therapy, an ECOG status 0-2, and adequate organ function. Each patient's MDS-related mutations are identified and autologous T cells are immunized ex vivo with peptides corresponding to the mutated protein(s), then expanded and suitability-tested for experimental infusion (referred to as PACTN). Importantly, the T cells must demonstrate neoantigen specificity and must kill autologous MDS stem-progenitor cells prior to qualification for infusion. Each eligible subject receives a single infusion of autologous PACTN followed by intensive monitoring for adverse events (AEs) for 4 weeks and periodic monitoring for 1 year. The primary study end-point (EP) is assessment of dose-limiting toxicity (DLT) and maximum tolerated PACTN dose (MTD). Secondary EPs include disease response 1 month after PACTN infusion, overall and progression-free survival at 6 and 12 months, and assessment of the peak abundance and persistence of the infused T cells in peripheral blood. Exploratory EPs include an assessment of the effect of PACTN infusion on the allele frequencies of the targeted and non-targeted MDS mutations in blood and marrow leukocytes. Results: At this time, two subjects have been infused with PACTN in the first dose cohort. Neither subject had an infusion reaction, severe AE, or DLT after follow-up for 2-3 months, nor has a disease response occurred in these subjects. Of interest, the infused PACTN product in the first subject showed 59% clonal dominance by a single T cell receptor (TCR) clone that was present at only 0.002% in patient's blood prior to T cell immunization with neoantigen related peptides. Multiple additional expanded TCR clones were also identified in the infused PACTN product. The presence of a dominant TCR clone in the PACTN product enabled the assessment of the in vivo abundance and persistence of the clone after PACTN infusion. The dominant clone expanded between day 1 and day+4 after PACTN infusion to a peak frequency of 0.13%, representing a 64-fold expansion of this TCR compared with the pre-infusion sample of blood leukocytes, then decreased to 0.09% by day +8. The clone was also demonstrated in bone marrow on day +15 at a frequency of 0.03%, representing a 20-fold expansion of this TCR clone compared with the pre-infusion marrow sample. Similar studies on the second subject are in progress, and will be continued in future subjects as the clinical trial continues. Finally, our studies show that it has been possible to effectively immunize autologous T cells to patient-specific neoantigens in all patients studied with MDS (n=4) and also all patients with AML (n=3) studied to date. Conclusion: The early results of this clinical trial support the feasibility and safety of this novel approach to adoptive T cell mediated immunotherapy for patients with higher-risk MDS and encourages continuation of the trial in the higher dose level cohorts. Disclosures Ferrari: Persimmune, Inc.: Employment. Tarke:Persimmune, Inc.: Employment. Fields:Persimmune, Inc.: Employment. Ferrari:Persimmune, Inc.: Employment. Ni:Persimmune, Inc.: Employment. Ferrari:Persimmune, Inc.: Employment. Warner:Persimmune, Inc.: Employment. Jochelson:PersImmune, Inc.: Consultancy. Bejar:Celgene: Consultancy, Honoraria; AbbVie/Genentech: Consultancy, Honoraria; Takeda: Research Funding; Genoptix: Consultancy; Modus Outcomes: Consultancy; Foundation Medicine: Consultancy; Astex/Otsuka: Consultancy, Honoraria. Vitiello:Persimmune, Inc.: Employment. Lane:PersImmune, Inc.: Employment.

Description: Background: Myelodysplastic Syndromes (MDS) are a heterogeneous hematologic malignancy characterized by bone marrow failure and cytopenias. The median survival rate for patients with higher-risk MDS who fail standard-of-care chemotherapy with hypomethylating agents (HMAs) is less than 6 months, and the only curative treatment for these patients is hematopoietic stem cell transplant (HSCT). Over the past 10 years, immunotherapy as a cancer treatment has achieved variable levels of success in different tumor types. There are currently 22 active clinical trials of immunotherapies for MDS (www.clinicaltrials.gov; 7/30/19), including our phase I clinical trial with a personalized adoptive cellular therapy targeting MDS patient neoantigens (NCT 03258359). Because MDS patients are frequently monocytopenic and the existing literature is inconsistent regarding the ability of MDS patients' monocytes to support T cell activation, we compared the activation of MDS T cells with those of healthy donors in the presence of autologous monocytes. Methods: Peripheral blood mononuclear cells (PBMC) from 5 healthy donors and 7 higher-risk MDS patients were cryopreserved after Ficoll separation. These PBMC were thawed and aliquoted into 6 replicate wells of 200,000 cells in 96-well u-bottom plates in R-10 culture medium. Half of the wells were treated with 25 ng/mL OKT3 and 200 U/mL IL-2. After 48 hours at 37˚C with 5% CO2, the wells were collected for analysis by flow cytometry. Beads were used to detect T cell activation induced secretion of IFNg, TNFa, IL-4, IL-10, and IL-17 in the supernatant and fluorescent antibodies were used to phenotype viable cells for CD3, CD4, CD8, and the T cell activation markers, CD69, CD25, CTLA-4, PD-1, and HLA-DR. Results: We measured a higher release of IFNg and TNFa in donor PBMC compared to MDS patients after OKT3/IL-2 activation, p 〈 0.01 and 0.04, respectively by 2-way ANOVA. The expression of CD69, CD25, HLA-DR, and CTLA4 increased variably on activated T cells from donors or MDS patients, but expression of CD4+CD25+ was more frequent on donor T cells after activation (p = 0.03). Activation also resulted in a higher frequency of PD-1 expression on donor CD4+ and CD8+ T cells than on MDS T cells (p 〈 0.01 and 〈 0.01, respectively). Interestingly, on both MDS and normal T cells the percentage of CD8+PD1+ activated cells correlated strongly with the percent of CD14+ monocytes present in the PBMC (R2 = 0.92 and 0.60 respectively; Fig 1a and 1b). We designed further experiments to test whether this was a patient intrinsic phenomenon, or if the absolute number of CD14+ monocytes in the PBMC was associated with different levels of PD1 expression upon T cell activation. First, we separated CD14+ cells from the PBMC of a patient with MDS using magnetic beads. Then CD14+ cells were added back to the CD14-depleted PBMC at a final percent of 0.5, 5, 10, 20, 35, 70, or 100% of the original amount. Unmodified PBMC was included as a control and all cells were stimulated with OKT3 and IL-2 or left in R-10 medium without stimulus. After 24, 48, and 70 hours, samples were collected to analyze by flow cytometry for CD3, CD4, CD8, CD14, and PD1 expression. The results show that an increasing percent of monocytes corresponded to the increased expression of PD1 on CD8+ and CD4+ T cells. Conclusion: Our results show that there are variable reductions in markers of T cell activation and cytokine secretion in MDS patients compared to healthy donors. We also observed that the fold increase in activation induced PD-1 expression was well correlated with the percent of CD14+ monocytes in the PBMC of both MDS patients and healthy donors. Direct experimentation revealed that this correlation is a cause-effect relationship. We are continuing to investigate the role of monocytes in T cell activation in MDS patients. Disclosures Bejar: Celgene: Consultancy; Takeda Pharmaceuticals: Research Funding; AbbVie/Genentech: Consultancy, Honoraria; Astex/Otsuka: Consultancy; Modus Outcomes: Consultancy; Daiichi-Sankyo: Consultancy. Lane:PersImmune, Inc.: Employment.

Description: Background: Myelodysplastic syndromes (MDS) have been associated with alterations in the bone marrow microenvironment and include abnormal innate and adaptive immune responses. The extent to which these changes contribute to the pathogenesis of MDS is unclear. The heterogeneous nature of MDS makes it difficult to distinguish changes in intrinsic cell function for microenvironmental alterations in the mutated myeloid compartment. T cells, which are in the lymphoid compartment and less frequently contain MDS mutations, can potentially be used to evaluate immune dysfunction in the presence of malignant myeloid cells. Here we examine the immunophenotypes and proliferative function of T lymphocyte and monocyte populations from higher-risk MDS patients. Methods: Peripheral blood mononuclear cells (PBMC) from patients with IPSS-R Intermediate/High/Very High-risk MDS (herein referred to collectively as higher-risk MDS) and age-matched healthy donors (n = 15 and n = 12, respectively) were polyclonally stimulated with an antibody against CD3 (OKT3) in the presence of IL-2 (50 U/mL). Phenotypic analysis was performed at baseline and 46 hours after the addition of OKT3 and IL-2. T cell proliferation was measured by staining PBMC with proliferation dye (CellTrace Violet) and measuring fold proliferation after eight days in culture. Proliferation statistical comparisons were made by unpaired t-test. Phenotypic parameters were measured by flow cytometry and statistical analysis performed with the Kruskal-Wallis one-way analysis. Results: As previously reported, higher-risk MDS patient T cells had decreased capacity for proliferation when compared to healthy donor counterparts (Figure 1A p = 0.0096). This difference in proliferative capacity was not associated with either the level of PD-1 on the surface of T cells (r = 0.2956) or the level of PD-L1 on monocytes (r = 0.3859) in stimulated PBMCs. Patients with higher-risk MDS tended to have lower PD-L1 surface expression on monocytes compared to healthy donor counterparts, albeit not significantly (Figure 1C p = 0.1). However, PD-1 expression on T cells was comparable between groups both before and after stimulation (Figure 1B CD4 cells: p = 0.58; CD8 cells p = 0.58). This suggests that T cell proliferation from patients with higher-risk MDS might not be inhibited through the PD-1/PD-L1 axis. Of note, higher-risk MDS patient monocytes expressed less CD80 and HLA-DR both pre- and post-stimulation when compared to healthy donors (Figure 1C CD80: p = 0.0001 and 0.037; DR: p = 0.012 and 0.019, respectively), implying a potential dysfunction in the monocytic stimulatory and antigen presentation capacity. PD-L1 surface expression is low at baseline (an average of