Academic literature on the topic 'Red Bull Cocktails'

Abstract Four CML patients were investigated for post transplant chimerism for total marrow (BM) cells and Mesenchymal Stem Cell (MSC) populations purified and propagated from BM cells. Patients were conditioned with BuFluATG and after allogeneic PBPCT were disease free with proven hematological, cytogenetical and genetical remission. For the study 15–30 ml fresh BM aspirates were phenotypically characterized for the presence of cell populations which may contain MSC. We found (median values): 11.7% CD45-CD34− 1.7% CD45-CD34-CD105+, 0.13% CD45-CD34-CD90+ and 0.03% CD45-CD34-CD73+ cells. These BM populations contained 0.75% CD34+ cells. Genetical work showed that BM cells were BCR-ABL negative and their STR informative allele patterns were consistent with those of donors. BM cells were processed as follows: incubation with Glycophorin A, CD3, CD14, CD19, CD66b, CD38 antibody cocktail which by cross-linking unwanted cells with red blood cells led to rosette formation (RosetteSep MSC Enrichment Cocktail, StemCell Technology), unrosetted cells (MSC enriched) were recovered from the interface after buoyant gradient centrifugation and contained (median values): 68.7% CD45-CD34-, 21.6% CD45-CD34-CD105+, 1.1% CD45-CD34-CD90+ and 0.4% CD45-CD34-CD73+. MSC enriched BM populations were cultured in Medium for Human MSC with Stimulatory Supplements (StemCell Technology). After 10–14 days CFU-F colonies were scored (median value was 77 CFU-F/106 cells) and cells were further cultured until >90% confluence of fibroblast-like cells were reached (usually 3 to 4 weeks after culture initiation). The cells were detached with 0.05% trypsin-EDTA and studied for STR allele patterns, the presence of BCR-ABL transcripts (at that time cells showed STR alleles of the recipient pattern for the first time - mixed chimerism) and the bulk of cells were further passaged. Usually after 3–4 passages (within 7–8 weeks) when fibroblast-like stromal cell populations reached the level of 3x106 cells, cultures were terminated and the cells were studied. These cells were in (median values) 27.0% CD45-CD34-, 23.8% CD45-CD34-CD105+, 26.5% CD45-CD34-CD90+ and 24.3% CD45-CD34-CD73+. The cells had a fibroblast-like morphology but only 26% had phenotype features of MSC on average. Therefore, the population consisted of MSC and more differentiated cells originated from CFU-F (MSC). RNA and DNA were isolated from the cells propagated for 7–8 weeks from the MSC enriched BM populations were BCR-ABL negative. However, their STR informative allele patterns were consistent with those of the recipients in variance to primary BM cell populations which was in all 4 cases of donor origin. Conclusions: with the use of the RosetteSep MSC enrichment purification system BM cells can be enriched in CFU-F which paralleled with an increase in the proportion of CD45-CD34-, CD45-CD34-CD90+, CD45-CD34-CD73+ and CD45-CD34-CD105+ cells, CFU-F BM enriched populations can be cultured with Medium for Human MSC with Stimulatory Supplements for successful propagation of fibroblast-like cells with kinetics documenting ex potential growth after 38 days (median) of culture, Cells originated from CFU-F were in contrast to the BM hematopoietic compartment of the recipients origin and were also BCR-ABL negative, MSC were not replaced by allogeneic PBPC-graft derived MSC.

BackgroundType I interferon (IFN) plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE) and dermatomyositis (DM)/polymyositis (PM) as typically evidenced by upregulation of type I IFN-stimulated genes (ISGs) in peripheral immune cells [1]. In addition, a distinct monocyte cytokine signature, which was induced by plasma from pediatric SLE patients and abrogated by Janus kinase (JAK) inhibition, was reported [2]. However, contribution of serum factors to expression of ISGs was not fully elucidated.ObjectivesTo investigate the cytokine induction profile in multiple myeloid lineages by serum from patients with SLE or DM/PM using the whole blood stimulation system and identify the signaling pathway relavant to the monocyte cytokine signature.MethodsSerum was collected from newly diagnosed, untreated, and clinically active adult patients with SLE (SLE group), anti-MDA5 antibody-positive DM (MDA5 group), and anti-aminoacyl-tRNA synthetase (ARS) antibody-positive DM/PM (ARS group), and healthy controls (HCs) (n= 10 for each group). Heparinized whole blood from healthy donors was incubated with control serum (10 v/v%), patient serum (10 v/v%), or IFN-β in the presence of protein transport inhibitor cocktail for 6 hours. Red blood cells were lysed, and then leucocytes were fixed at a single step. Intracellular staining was performed and expression of 11 cytokines in CD14+monocytes, CD1c+dendritic cells (DCs), and CD123+DCs were analyzed using flow cytometry. A cut-off level was determined by 2% positivity of each cytokine in unstimulated condition. For transcriptomic analyses, sorted CD14+monocytes from healthy donors were incubated with serum (SLE, MDA5, ARS, or HCs), or IFN-β (n= 3 for each group) for 4 hours, and bulk RNA-sequencing was performed using the Illumina NextSeq 500 platform. RNA-seq raw sequence reads analysis and pathway analysis were performed using the Strand NGS software. To evaluate significance of JAK-signal transducer and activator of transcription (STAT) pathway, whole blood from healthy donors was pre-incubated with upadacitinib, a JAK1 inhibitor, stimulated with patient serum for 6 hours, and analyzed for cytokine expression as described above.ResultsSerum from SLE and MDA5 groups induced significantly higher monocyte chemoattractant protein-1 (MCP1) and interleukin-1 receptor antagonist (IL-1RA) expression in CD14+monocytes than serum from HCs (Figure 1). These monocyte cytokine signatures were closely resembled that induced by IFN-β stimulation. Serum from ARS group did not induce any significant cytokine expression in CD14+monocytes. No significant cytokine expression was observed in CD1c+DCs or CD123+DCs. RNA-seq demonstrated that 612 and 462 genes were upregulated (fold change >2 relative to control) following stimulation with serum from SLE and MDA5 groups, respectively. In these upregulated genes, 383 genes were commonly upregulated across SLE and MDA5 groups and IFN-β. Pathway analysis revealed similar transcriptional profiles in SLE and MDA5 groups: upregulated genes were most frequently involved in IFN-αβ signaling pathway including multiple ISGs and STAT1/2. Upadacitinib significantly abrogated the monocyte cytokine signature, and MCP1 and IL-1RA expression induced by serum from SLE and MDA5 groups in a dose-dependent manner (p< 0.001 in all analyses).ConclusionThese results suggest that serum factors in patients with active SLE and anti-MDA5 antibody-positive DM can induce shared monocyte cytokine signature through type I IFN pathway. CD14+monocytes ‘primed’ by serum might contribute to the pathogenesis of these diseases.References[1]Rheumatology (Oxford).2017;56:1662[2]J Autoimmun. 2017;81:74Figure 1.MCP1 (A) and IL-1RA (B) expression in CD14+monocytes induced by serum from SLE and anti-MDA5 antibody-positive DM.Horizontal bars represent median values.P-values were calculated using Kruskal-Wallis test followed by Dunn’s multiple comparisons test.Acknowledgements:NIL.Disclosure of InterestsShohei Nakamura: None declared, Yuko Okamoto: None declared, Yasuhiro Katsumata Speakers bureau: GlaxoSmithKline K.K., AstraZeneca K.K., Sanofi K.K., Pfizer Japan Inc., Janssen Pharmaceutical K.K., Chugai Pharmaceutical Co., Ltd., Asahi Kasei Pharma, Astellas Pharma Inc., Mitsubishi Tanabe Pharma Corporation, Masayoshi Harigai: None declared.

Abstract Background: Chronic myelomonocytic leukemia (CMML) is an aging disease that arises on the background of clonal hematopoiesis, commonly involving multiple TET2 mutations or TET2/SRSF2 co-mutations. In a prior study of 1084 CMML patients (Leukemia 2020; 34:1407-1421), we demonstrated a high prevalence of TET2 mutations (56%) in CMML, with approximately 30% of patients having &gt;1 TET2 mutation. The cell-type and allele specific distribution of these mutations remains of great interest and are thus far poorly defined. To visualize the genotypic and immunophenotypic correlates, we performed single-cell DNA analysis and simultaneous protein profiling on 2 CMML patients with multiple TET2 mutations. Methods: After Mayo Clinic IRB approval, two CMML patients with dual TET2 mutations and available peripheral blood mononuclear cells were included in the study. Patient samples were labeled with TotalSeq TM -D Heme Oncology Cocktail (BioLegend, San Diego, CA) consisting of 45 unique oligo-conjugated cell surface markers. The samples were then washed and loaded into a Tapestri microfluidics cartridge, where single cells (3,500 cells/µL) were encapsulated, lysed and barcoded using the MissionBio Myeloid custom amplicon panel consisting of 312 amplicons in 45 genes relevant to myeloid disorders (MissionBio, San Francisco, CA). The individual barcoded DNA and protein for each cell was purified, recovered, and labeled with Illumina i5/i7 indices, quanitifed by Agilent Bioanalyzer and pooled for sequencing. The libraries were sequenced via Illumina NovaSeq by the Mayo Genomics Core, and data was recovered and analyzed using MissionBio Tapestri Insights v3.0.2. Results: Two dual TET2 mutated CMML patients were included in the study, both male, ages 79 and 83, and both with CMML-0. Case 1 had TET2 p.G1172Efs*53 (variant allele fraction/VAF-6%) and p.H1732Tfs*13 (VAF-13%) mutations, while case 2 had TET2 p.Y620Ffs* (VAF-85%), p.G898* (VAF-82%), and SRSF2 p.95H (VAF-45%) mutations, respectively. In both patients, TET2 mutations were found to be preferentially distributed in monocytes (CD11b+, CD14+, CD64+, CD13+, CD33+, HLA-DR+), with very few classically defined granulocyte (CD16+,CD13+.CD33+, HLA-DR neg), B (CD19+,CD22+)/T (CD2+,CD4+,CD5+,CD7+) lymphocytes, NK cells (CD16+/CD56+/HLA-DR neg) and plasma cells (CD38+/CD138+), demonstrating mutant TET2. Among monocytes, TET2 mutations were enriched in classical/M01 (CD11b+, CD14+,CD16-) and intermediate/M02 monocytes and were infrequent in the MO3 fraction, commensurate with monocyte repartitioning seen in CMML. Interestingly, a large number of single cells with TET2 mutations also demonstrated an aberrant surface phenotype, with universal expression of CD14+ and CD11b+, along with a mixture of other markers (CD163+, CD11c, CD4+, CD2+, and/or CD56+). With regards to allelic distribution, while several permutations and combination were seen involving heterozygosity and homozygosity for the TET2 mutations identified on bulk sequencing, data was corroborative for both TET2 mutations being located on trans alleles. Conclusions: In this study, we demonstrate that TET2 mutations in CMML are highly enriched in classical circulating monocytes and infrequently occur in classical granulocytes, B/T and NK cells; with multiple TET2 mutations most commonly co-existing on trans alleles. Analyses on additional samples along with copy number variation assessments are ongoing. Figure 1: Lollipop plot of TET2 exons with dual TET2 mutations labeled above and oxygenase domain labeled in green; below are number of cells identified with each genotype for Case 1 (A) and Case 2 (B). Mutations are labeled proximal (yellow) and distal (red) and alleles are in grey. The directionality of the arrows to different genotypes is based on decreasing cell number and genotype. Uniform manifold approximation and projection (UMAP) plot of Case 1 (C) and Case 2 (D) with cells clustered by immunophenotype and labeled by genotype (non TET-2 mutated purple, heterozygous TET2 in green, homozygous TET2 in yellow). Relative protein expression of T-cell phenotype (CD14-, CD3+, CD4+/-,CD8+/-, CD5+, CD7+, CD2+), or classical monocytes (CD14+/CD16-) for Case 1 (E & F) and Case 2 (G & H) overlayed on UMAP. Royal blue represents non TET2-mutated cells, and mutated cells are labeled in pastel blue, green and light orange by mutation type. Figure 1 Figure 1. Disclosures Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN) and myelodysplasia (MDS), identified mutations with different allele frequencies. Recent studies of clonal hematopoiesis (CH) discovered a subset of MM disease alleles, while other alleles are only observed in overt MM. These observations suggest an important pathogenetic role for the chronology of mutational acquisition. Although bulk sequencing informs prognostication, it cannot distinguish which mutations occur in the same clone and cannot offer definitive evidence of mutational order. Delineation of clonal architecture at the single cell level is key to understanding how the sequential/parallel acquisition of somatic mutations contributes to myeloid transformation. In order to elucidate the clonal structure of MM, we designed a custom single cell 109 amplicon panel of the most frequently mutated amplicons in 50 MM genes using the Mission Bio Tapestri v2 platform. Viable cells were sorted from 90 samples from 78 patients with CH, AML, and MPN/post-MPN AML followed by single cell amplification/sequencing. Mutation calls were filtered based on read depth, quality, and alleles genotyped per cell. We reconstructed a random distribution of clones by permuting genotype calls across cells and generated empirical p values for each clone. To identify dominant clones, we used a Poisson test to determine clones were significantly enriched compared to the mean clone size. Clones with significant p-values (p &lt;0.05) were used to generate plots of clonal architecture of each sample (Figure 1A). Despite significant clonal complexity, the majority of MM patients (80%;72/90) present with one (51/90; 56.7%) or two (21/90; 23.3%) dominant clones. These data show there are specific genotypic combinations which lead to clonal dominance with increased fitness relative to other clones and/or suppression of minor clones by dominant clone(s). We next investigated whether specific molecularly defined AML subtypes had increased clonal complexity. FLT3-ITD mutant AML samples had a significantly greater number of clones (p &lt; 0.002) compared to AML samples with multiple epigenetic modifier mutations. Similar findings were not observed when comparing AML samples with epigenetic mutations to RAS pathway mutant samples. We next investigated whether specific mutations were likely to co-occur/be mutually exclusive at a single cell level. We observed evidence of oligoclonality in CH, including parallel acquisition of DNMT3A mutations and clones with multiple mutations in the absence of progression to MM. By contrast, in MM the dominant clone(s) almost always harbored multiple epigenetic modifier mutations, suggesting cooperative epigenetic remodeling in myeloid transformation. Mutations in signaling effectors (FLT3-ITD/TKD; RAS/RAS) were mutually exclusive. We observed distinct FLT3-mutant clones in FLT3-mutant AML patients and parallel acquisition of different RAS pathway mutations. We used this data to develop clonal architecture trees in each patient, giving us a definitive picture of mutational acquisition and transformation at a single cell level. We calculated a Shannon diversity score and observed an increase in clonal complexity with disease evolution; CH samples had the lowest clonal diversity and FLT3-ITD AML patients the highest clonal diversity (Figure 1B). We extended our findings by combining cell surface marker assessment and single cell mutational analysis. Patient samples were stained with an antibody cocktail of 6 oligo-conjugated antibodies with barcode tags prior to single cell sequencing, which allowed simultaneous acquisition of single cell immunophenotypic and genotypic data. This allows us to identify distinct populations of stem/progenitor cells with distinct clonal/mutational repertoires (Figure 1C). Additional data will be presented with this novel approach, which allows us to combine an assessment of stem/progenitor cell frequency with genetic data. This includes studies of CD34+ and CD34- AML, which show striking differences in mutational representation in different stem/progenitor compartments. In summary, our studies of clonal architecture at a single cell level provide us novel insights into the pathogenesis of myeloid transformation and give us new insights into how clonal complexity contributes to disease progression. Disclosures Ooi: Mission Bio: Employment, Equity Ownership. Mendez:Mission Bio: Employment, Equity Ownership. Carroll:Janssen Pharmaceuticals: Consultancy; Incyte: Research Funding; Astellas Pharmaceuticals: Research Funding. Papaemmanuil:Celgene: Research Funding. Viny:Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Levine:Roche: Consultancy, Research Funding; Amgen: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Gilead: Consultancy; Celgene: Consultancy, Research Funding.

BackgroundSarcoma is a group of rare bone and soft tissue tumors with over 50 distinct subtypes. Survival rate ranges widely due to the lack of efficacious treatments. Immunotherapy, such as adoptive cell therapy (ACT), has drawn significant interest due to its minimal toxicity. In ACT, tumor infiltrating lymphocytes (TILs) are isolated from patients, expanded, and autologously infused back. We recently observed TILs’ presence in Undifferentiated Pleomorphic Sarcoma (UPS) and Myxofibrosarcoma (MFS) tumors and found that tumor’s PD-L1 overexpression is correlated with better clinical outcome in UPS but not MFS.1 The Th1 anti-tumoral inflammatory pathway was highly activated in the former cohort, which may explain the favorable outcome. We hypothesize that there are phenotypic and functional differences between TILs of UPS with differential PD-L1 expression, which may be related to clinical outcomes. However, sarcoma TILs are rare and challenging to culture, which significantly impedes their studies. We first aim to robustly expand sarcoma TILs to sufficient numbers.MethodsTumors’ PD-L1 expression was determined by RT-qPCR (table 1). To initiate the tumor-fragment (TF) method of TIL culturing, primary tumors were fragmented and cultured in IMDM, IL-2, and 10% HSA. We further optimized the TF protocol to expand rare sarcoma TILs. Rapid expansion protocol (REP) with anti-CD3/anti-CD28 co-stimulating beads was employed for additional expansion. During REP, TILs were co-treated with gamma-chain cytokines (IL-2, 7, 15, 21).ResultsOf the 15 MFS TIL populations expanded, only 40% achieved sufficient growth (1x106) for analysis (figure 1A). Our optimized TF protocol expanded TILs from 8 UPS cases with a 62.5% success rate (figure 1B). UPS TILs were further stimulated with REP and various gamma-chain cytokine treatments. In ACT, prolonged culturing with IL-2 is known to cause activation-induced cell death, problematic in clinical treatments. We demonstrated that treatments with a Trio-cocktail (IL-7, 15, and 21) or IL-15 alone can achieve TIL proliferation comparable to that of IL-2 (figure 2).Abstract 168 Figure 1Initial TIL Culturing with Tumor Fragment Method. Figure A.. The traditional tumor fragment protocol was used to expand TILs of four MFS cases. TILs were cultured and expanded from primary tumor fragments in IL-2 (6000IU/mL) supplemented complete media (CM) over four weeks in duration. Fifteen TIL populations were derived from the four MFS cases. Populations were categorized based on their growth rates and labeled as ‘1’ or ‘2’ representing ‘fast’ or ‘slow’ growing TILs, respectively. Additional populations ‘A’ and ‘B’ represent biological replicates. Population TIL164 ‘2’ had no replicates. At Week 4, populations’ cell counts were determined via hemocytometer. As shown, only 6 out of 15 populations achieved > 1x10^6 cells (40% success rate). Figure B. An optimized tumor fragment protocol was used to expand TILs of eight UPS cases. Optimization includes shortening the culturing duration from four weeks to two weeks, reducing frequency of cell culture disruption, and adjusting cell culture environments. TILs were expanded from primary tumor fragments in CM over two weeks in duration. At Week 2, populations’ cell counts were determined via hemocytometer. As shown, 5 out of 8 cases achieved >1x10^6 cells (62.5% success rate).Abstract 168 Figure 2Gamma-chain cytokine treatments of UPS TILs. Gamma-chain cytokine treatments of UPS TILs with CD3/CD28 stimulation. Magnetic Dynabeads coated with CD3 and CD28 monoclonal antibodies were used to stimulate cells at a bead to cell ratio of 1:3. In ACT, IL-2 is a gold-standard cytokine that facilitates potent T-cell growth. However, it is known to cause activation-induced cell death. Resulting TIL population also possesses an exhausted-effector phenotype with low durability. UPS TIL Case 52 and Case 166 were treated with various interleukins during two weeks of REP, including gamma-chain IL-7, 15, 21 and inflammatory IL-12. IL-7, IL-12, and IL-21 individually did not elicit significant T-cell growth. IL-15 alone and in combination with IL-7 and IL-21 yield growth comparable to IL-2. IL-2 was obtained from Novartis (50ng/mL). All other cytokines were obtained from PeproTech (25ng/mL).Abstract 168 Table 1Tumor PD-L1 RNA Expression. Four MFS and eight UPS cases were processed. Tumors’ PD-L1 RNA expression was determined via RT-qPCR and evaluated as a ratio with the housekeeping gene STAM2. TIL359’s PD-L1 status has yet to be evaluated.ConclusionsSarcoma infiltrates are difficult to culture, and their roles remain largely unstudied. By optimizing the TF protocol in conjunction with anti-CD3/CD28 treatments, we developed a robust in vitro pipeline to expand rare sarcoma TILs, enabling downstream characterization. We also demonstrated the potential for alternate gamma-chain cytokines to favorably replace IL-2 during TIL expansion. Future phenotypic and functional evaluation of UPS TILs would elucidate the impact of tumors’ differential PD-L1 expression on UPS patients‘ prognoses. These findings would inform the implementation of ACT in sarcoma treatments.ReferencesWunder J, Lee M, Nam J, Lau B, Dickson B, Pinnaduwage D, Bull S, Ferguson P, Seto A, Gokgoz N, Andrulis I. Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation. Onco Immunology 2020;9: e1737385-1- e1737385-13.Ethics ApprovalPatients provided signed consent before study entry, as approved by Mount Sinai Hospital’s Ethics Board (REB#01-0138-U).