Collection Of Peripheral Blood Mononuclear Cells Research Articles

Technical pitfalls when collecting, cryopreserving, thawing, and stimulating human T-cells.

The collection, cryopreservation, thawing, and culture of peripheral blood mononuclear cells (PBMCs) can profoundly influence T cell viability and immunogenicity. Gold-standard PBMC processing protocols have been developed by the Office of HIV/AIDS Network Coordination (HANC); however, these protocols are not universally observed. Herein, we have explored the current literature assessing how technical variation during PBMC processing can influence cellular viability and T cell immunogenicity, noting inconsistent findings between many of these studies. Amid the mounting concerns over scientific replicability, there is growing acknowledgement that improved methodological rigour and transparent reporting is required to facilitate independent reproducibility. This review highlights that in human T cell studies, this entails adopting stringent standardised operating procedures (SOPs) for PBMC processing. We specifically propose the use of HANC's Cross-Network PBMC Processing SOP, when collecting and cryopreserving PBMCs, and the HANC member network International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) PBMC Thawing SOP when thawing PBMCs. These stringent and detailed protocols include comprehensive reporting procedures to document unavoidable technical variations, such as delayed processing times. Additionally, we make further standardisation and reporting recommendations to minimise and document variability during this critical experimental period. This review provides a detailed overview of the challenges inherent to a procedure often considered routine, highlighting the importance of carefully considering each aspect of SOPs for PBMC collection, cryopreservation, thawing, and culture to ensure accurate interpretation and comparison between studies.

Utilizing low‐volume PBMC samples from Alzheimer’s disease clinical research cohorts for immune cell RNA‐sequencing

AbstractBackgroundStudies using peripheral blood mononuclear cells (PBMCs) from individuals with Alzheimer’s disease (AD) biomarker information has the potential to clarify the role of peripheral immune systems in AD. However, PBMC collection often requires immediate labor‐intensive processing and large‐volume collection, making it difficult to implement in clinical AD research. Here, we present our optimized protocol to collect, cryopreserve, and process PBMC samples for monocyte and lymphocyte RNA‐sequencing (RNA‐Seq) studies in large clinical AD research programs.MethodWe used 8 mL Cell Preparation Tubes (CPT) to collect blood samples, and each CPT was processed and cryopreserved into two aliquots per the manufacturer’s protocol. Later, one PBMC vial ( = 4 mL blood) was thawed and washed in RPMI (+10% FBS, +1% PS). After recording cell numbers and viability, 2.0×106 cells from each sample were stained with anti‐CD4, CD8, CD14, CD20 antibodies, and Sytox blue (viability) in PBS (+2% FBS). The cells were washed and resuspended in 500 uL PBS (+2% FBS; target concentration 4.0×106/mL) for flow cytometry. We sorted and collected CD4+ T cells, CD8+ T cells, CD14+ monocytes, and CD20+ B cells into RNA lysis‐buffer (TCL + 1% BME), aiming for 5,000 or 10,000 cells/cell type/donor. We used the RNeasy micro kit for RNA extraction and Fragment Analyzer for quality control.ResultWe collected and cryopreserved PBMC samples from 436 Memory and Aging Cohort (MAC) participants. We used 28 samples for this pilot study (mean age = 68.2 ± 8.1, 21 females). From one cryopreserved aliquot ( = 4 mL of blood), we recovered an average of 3.40×106 ± 1.43×106 viable cells (mean viability 90.4% ± 5.8). Average quantity of RNA was 12.2 ± 4.95 ng/104 cells. The RNA integrity (RIN) score was 8.89 ± 1.7, consistent with excellent RNA quality (Fig. 1).ConclusionOur low‐volume protocol (4 mL of blood per participant) deployed in a large clinical AD study yielded excellent‐quality RNA for cell‐type‐specific bulk RNA‐Seq (using SMART‐Seq2 library). This protocol can be readily adapted for cell‐type‐specific proteomics or single‐cell multiome projects and is a promising approach to deeply phenotype the peripheral immune system in the context of aging and AD.

A fully automated Lab-on-a-Disc platform integrated a high-speed triggered siphon valve for PBMCs extraction

Human Peripheral Blood Mononuclear Cells (PBMCs) are isolated from peripheral blood and identified as any blood cell with a round nucleus that exhibits immune responses and undergoes immunophenotypic changes upon exposure to various pathophysiological stimuli. Obtaining high-recovery and clinical-grade PBMCs without decreasing cell viability and causing stress is crucial for disease diagnosis and successful immunotherapy. However, traditional manual PBMCs extraction methods rely on manual intervention with less recovery rate and reliability. In this study, we introduced a novel and efficient strategy for the fully automated extraction of PBMCs based on a Lab-on-a-Disk (LoaD) platform. The centrifugal chip used percoll as density gradient media (DGM) for separation and extraction on account of the density difference of cells in whole blood, without labeling and any additional extra cellular filtration or cell lysis steps. Above all, we proposed a high-speed triggered siphon valve, which was closed under the speed of cell sedimentation and subsequently opened by increasing speed to complete the extraction of PBMCs. It can avoid the problem that previous siphon valves rely on unstable hydrophilic surface treatment and prime under low/zero speed conditions. With valves and the clock channel integrated on the chip, users can achieve fully automated collection of PBMCs. Compared with the clinical laboratory results, the recovery rate of extracted PBMCs was 80 %. The experimental results prove that the high-speed triggered siphon valve improves the extraction efficiency of PBMCs. The robust chips, which are not only simple to manufacture and assemble but also stable and reliable to use, have great potential in biomedical and clinical applications.

Transcriptomic profile of TNFhigh MAIT cells is linked to B cell response following SARS-CoV-2 vaccination.

Higher frequencies of mucosal-associated invariant T (MAIT) cells were associated with an increased adaptive response to mRNA BNT162b2 SARS-CoV-2 vaccine, however, the mechanistic insights into this relationship are unknown. In the present study, we hypothesized that the TNF response of MAIT cells supports B cell activation following SARS-CoV-2 immunization. To investigate the effects of repeated SARS-CoV-2 vaccinations on the peripheral blood mononuclear cells (PBMCs), we performed a longitudinal single cell (sc)RNA-seq and scTCR-seq analysis of SARS-CoV-2 vaccinated healthy adults with two doses of the Pfizer-BioNTech BNT162b2 mRNA vaccine. Collection of PBMCs was performed 1 day before, 3 and 17 days after prime vaccination, and 3 days and 3 months following vaccine boost. Based on scRNA/TCR-seq data related to regulatory signals induced by the vaccine, we used computational approaches for the functional pathway enrichment analysis (Reactome), dynamics of the effector cell-polarization (RNA Velocity and CellRank), and cell-cell communication (NicheNet). We identified MAIT cells as an important source of TNF across circulating lymphocytes in response to repeated SARS-CoV-2 BNT162b2 vaccination. The TNFhigh signature of MAIT cells was induced by the second administration of the vaccine. Notably, the increased TNF expression was associated with MAIT cell proliferation and efficient anti-SARS-CoV-2 antibody production. Finally, by decoding the ligand-receptor interactions and incorporating intracellular signaling, we predicted TNFhigh MAIT cell interplay with different B cell subsets. In specific, predicted TNF-mediated activation was selectively directed to conventional switched memory B cells, which are deputed to high-affinity long-term memory. Overall, our results indicate that SARS-CoV-2 BNT162b2 vaccination influences MAIT cell frequencies and their transcriptional effector profile with the potential to promote B cell activation. This research also provides a blueprint for the promising use of MAIT cells as cellular adjuvants in mRNA-based vaccines.

Chemical derivatization as a novel strategy for selective and sensitive determination of intracellular di-and triphosphate anabolites in peripheral blood mononuclear cells

Quantitation of intracellular tri-phosphate anabolite, GSK1-TP, was required to understand drug efficacy of a proprietary molecule, GSK1; while quantitation of the di-phosphate, GSK1-DP, provided an indicator of potential GSK1-TP instability during sample processing and storage. A novel derivatization approach with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and hexylamine was developed to mitigate the challenges associated with the analysis of GSK1-DP and GSK1-TP, rendering them more amendable to reversed-phase LC-MS/MS detection. Extensive effort was spent to minimize the analyte loss and cell counts variability during peripheral blood mononuclear cells (PBMC) collection and best practices were thoroughly discussed. A solution of 30/70/2 (v/v/v) RPMI-1640/methanol/trichloroacetic acid proved to be an effective method of analyte stabilization and recovery. Methods were developed for simultaneous analysis of GSK1-DP and GSK1-TP with a limit of quantitation of 2.0 ng/mL in dog and rat PBMC lysate, with a subsequent improvement to 0.1 ng/mL for the analysis of GSK1-TP in human PBMC lysate. All three assays were found to be robust over the PBMC concentration range of 1–25 × 106 lysed cells/mL. This novel methodology could alleviate some challenges associated with the bioanalysis of intracellular phosphorylated anabolites such as PBMC collection variability, analyte instability, poor chromatographic retention, high carryover, matrix effect and insufficient assay sensitivity.

Limited Sequence Variation and Similar Phenotypic Characteristics of HIV-1 Subtype C Gag Variants Derived From the Reservoir and Pre-Therapy Plasma

HIV variants present in the reservoir, particularly in tissues, may differ from those present in peripheral blood prior to therapy initiation, and characterisation of these reservoir variants could better inform immune-based interventions for HIV cure. In the present study, Gag sequence differences between variants derived from the lymph node and peripheral blood mononuclear cell (PBMC) reservoirs as well as those derived from pre-therapy plasma, were investigated in 24 HIV-1 subtype C-infected individuals. HIVgagamplification was successful for 20 individuals, where 4 were controls including one untreated individual and 3 early treated individuals with LN collection within 2 weeks of treatment initiation. The remaining 16 individuals with LN and PBMC collection > 3 months after treatment initiation (median = 665 days), were further characterised. Recombinant viruses encoding patient-derived Gag-protease sequences from the pre-therapy plasma, LN reservoir, and PBMC reservoir, were constructed and the replication-competent viruses that grewin vitrowere used to further investigate whether there are specific features of Gag reservoir variants that may have relevance for strategies to cure HIV. Virus characteristics measured included replication capacity, interferon-alpha resistance, cell-to-cell spread ability, and induction of antiviral cytokines. A limited number of novel Gag mutations (median = 4) in the reservoir of 3/7 early treated participants and 9/9 late treated participants were observed, where the majority of these mutations were likely cytotoxic T lymphocyte (CTL)-driven and 48% were represented in the replication-competent viruses. The reservoir variants had very few unique potential CTL escape mutations (median = 3) in Gag compared to the number of these Gag mutations that were already present in the plasma-derived virus (median = 23) at the time of treatment initiation, which was similar whether treatment was initiated late or early. The data suggest that the extent of CTL escape in Gag overall is likely similar between early and late treated individuals as well as between the reservoir and pre-therapy variants. The sequence differences in Gag that were unique to the reservoir viruses did not result in significantly altered virus characteristics overall, and are therefore unlikely to affect effectiveness of immune-based interventions for virus eradication.

Phase 1 Study of Elotuzumab and Peripheral Blood Mononuclear Cell Reconstitution in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Phase 1 Study of Elotuzumab and Peripheral Blood Mononuclear Cell Reconstitution in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Detection of MYD88 L265P Mutation By Next Generation Deep Sequencing in Peripheral Blood Mononuclear Cells of Waldenstrom Macroguloblinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Introduction: Whole genome sequencing has recently revealed MYD88 L265P somatic mutations in Waldenstrom macroguloblinemia (WM) tumor cells. Allele-specific polymerase chain reaction (AS-PCR) using bone marrow (BM) aspirate and peripheral blood (PB) is the most frequently used technique to detect the specific mutation. In search for a non-invasive, simple, and more sensitive detection of MYD88 L265P mutation, we performed next generation deep sequencing (NGS) technique using unselected peripheral blood mononuclear cells (PBMCs) collected from WM and IgM monoclonal gammopathy of undetermined significance (IgM MGUS) patients. The main purpose of this study is to assess the clinical implication of MYD88 L265P mutation burden detected by NGS technique using unselected PBMCs.Patients and methods: The study was approved from the Institutional Review Board. After informed consent was obtained in accordance with the Declaration of Helsinki, PBMCs were collected from 38 patients with WM and 20 patients with IgM MGUS. The median time from diagnosis to PBMC collection was 2.2 years (range, 0-25 years). Twenty-nine (76.3%) of WM patients were previously treated. The overall response (CR+PR+MR) rate at the time of analysis was 75.8% (22/29). Genomic DNA was extracted from unselected PBMCs and 300 ng of DNA was used for NGS analysis. Variation rates of MYD88 at the nucleotide position corresponding to L265P were measured with an Illumina MiSeq DNA sequencer by molecular barcoding technology essentially as previously reported (Nature Protocols 12, 664:2017). PCR products were sequenced on a MiSeq DNA sequencer with a Reagent kit v2. for 300 cycles. Serial dilution assessment was carried out to investigate the sensitivity of the method by analyzing the DNA of BM sample from a MYD88 mutation positive patient diluted in DNA from a healthy control.Results: Dilution assessment demonstrated that NGS technique was detectable to a sensitivity of 0.02%, ×5 higher than previously reported sensitivity by AS-PCR. MYD88 L265P mutation was detected from 6/9 (66.7%), 8/29 (27.6%), and 11/20 (55%) in patients with untreated WM, previously treated WM, and IgM MGUS, respectively. Among the patients who were positive forMYD88 L265P mutations, the median percentage of mutant allele relative to WT was 3.0% (0.14-32.3%), 0.24% (0.02-33.8%), and 0.55% (0.06-2.85%), in untreated WM, previously treated WM, and IgM MGUS, respectively. Patients with Untreated WM and those with lymphadenopathy showed a trend toward higher mutant allele burden in PBMCs, but the difference was not statistically significant. Among 26 previously treated WM patients, those with at least minimal response showed significantly lower detection rate of MYD88 L265P mutation (13.6 vs. 75.0%, P=0.02) and mutant allele burden (median, 0.00% vs. 0.23%, P=0.01), compared to patients with less than minimal response.Conclusion: This study is the first to report NGS analysis of MYD88 L265P mutation using unselected PBMCs of WM and IgM MGUS patients. The sensitivity for MYD88 L265P mutation in unselected PBMCs from IgM MGUS patients was comparable with that detected by AS-PCR using CD19-selected PB cells (Leukemia, 2014;28:1698-704). Although its specificity is in need for further investigations, NGS technique successfully detected the mutation carried by minimal tumor cells included in unselected PBMCs with high sensitivity as AS-PCR using CD19-selected PB cells. The mutation detection rate of previously treated WM patients was lower than that of untreated patients in our study and previously reported cohort; however, this in turn may potentially indicate that NGS detection of MYD88 L265P mutation in PBMC be clinically utilized in assessing treatment response by monitoring tumor burden during patients' clinical course. DisclosuresNo relevant conflicts of interest to declare.

Phase 1 study of elotuzumab in combination with autologous stem cell transplantation and lenalidomide maintenance for multiple myeloma.

8021 Background: Elotuzumab is a humanized monoclonal antibody directed against SLAMF7 that is approved for use in relapsed multiple myeloma. We initiated a single-center, open label, phase 1 trial based on the hypothesis that the addition of elotuzumab and autologous peripheral blood mononuclear cell (PBMC) reconstitution to standard-of-care autologous hematopoietic stem cell transplantation (auto-SCT) and lenalidomide maintenance for consolidation therapy in myeloma patients will be safe and feasible. Methods: This is a Phase 1b, open-label, trial investigating elotuzumab and autologous PBMC reconstitution with auto-SCT consolidation therapy and lenalidomide maintenance. The primary objective of this study is to assess the safety and tolerability of elotuzumab and autologous PBMC reconstitution in the setting of auto-SCT and lenalidomide maintenance. The secondary objectives are to assess myeloma disease status and progression-free survival (PFS) after one year of treatment. Subjects must be eligible for auto-SCT, and meet inclusion/exclusion criteria. Fifteen subjects participated in this study. The treatment plan is: In addition to PBSC harvest, subjects undergo steady-state leukopheresis for PBMC collection. Subjects receive standard melphalan (day -1) and autologous stem cell rescue (day 0). Autologous PBMC are reinfused on day +3 and cycle 1 of elotuzumab 20 mg/kg IV is given on day +4. Subjects receive elotuzumab every 28 days up to cycle 12. Lenalidomide maintenance at 10 mg orally daily begins with cycle 4 of elotuzumab. The evaluable population constitutes all subjects who received at least four of the first five planned doses of elotuzumab. Results: 15 subjects have been enrolled in the study. All of these subjects are included in the safety population, having received at least 1 dose of elotuzumab. Nine of 15 subjects have completed 4 of the first 5 planned elotuzumab infusions.. The majority of adverse events, including infusion reactions attributable to elotuzumab, have been grade 2 or lower. There were no delays in hematopoietic reconstitution. No AEs were attributed to PBMC reconstitution. Conclusions: The combination of elotuzumab and PBMC reconstitution with standard auto-SCT and lenalidomide maintenance for consolidation therapy appears to be safe and feasible. The trial is ongoing and has completed accrual. The clinical results will be updated for presentation.

Comparison of Chimeric Antigen Receptor T Cells from Allogenic or Autologous Sources in Patients with Acute Lymphoblastic Leukemia

Introduction: The source of chimeric antigen receptor (CAR) T cells can be autologous or allogeneic, depending on the history of allogeneic hematopoietic stem cell transplantation (allo-HSCT). For patients with a history of allo-HSCT, CAR T cells are allogeneic, which can be manufactured from donors' peripheral blood mononuclear cells (PBMC) (donor-derived allogeneic CAR T cells, DD-alloCAR) or patients' own PBMC (recipient-derived allogeneic CAR T cells, RD-alloCAR). Unlike autoCAR T cells, which are activated by CAR only, alloCAR T cells receive activation signals from both T-cell receptor and CAR. The difference could affect CAR T-cell biology, resulting in different clinical outcomes.Methods: We retrospectively reviewed 31 consecutive patients who received CAR T-cell therapy in our center. Patients were divided into three groups, as autoCAR group, DD-alloCAR group and RD-alloCAR group. We compared the efficacy and safety profiles among the three groups. We also performed a subgroup analysis of patients received alloCAR.Results: Altogether there were 17 patients in the autoCAR, 11 in the RD-alloCAR and 3 in the DD-alloCAR groups. After a median follow-up of 9 months, the CR rate was 88.2% (95% CI 63.6-98.5%) in the autoCAR group and 100% (95% CI 71.5-100%) in the RD-alloCAR group, which had no significant difference (p=0.73). CR rate in DD-alloCAR was 66.7% (95% CI 9.43-99.1%). The median peak expansion of CAR T cells in the autoCAR was significantly higher than the RD-alloCAR group (p=0.007, Figure A). However, the median OS and EFS did not differ significantly among the groups. As far as adverse effects, RD-alloCAR group had significantly less patients with severe CRS (defined as Grade ≥ 3) than the autoCAR group (p=0.049), with significantly lower peak IL-6 level (p=0.021, Figure B). Neurotoxicity and the degree of cytopenia did not have significant difference among the groups. Acute GVHD occurred in 2 (18.2%) RD-alloCAR patients and 1 (33.3%) DD-alloCAR patients following CAR T-cell infusion. Univariate subgroup analysis of the patients who received alloCAR T-cell therapy showed the presence of cGVHD at the time of PBMC collection was significantly associated with less 6-month relapses (p=0.022); the median RFS in the no-cGVHD group was 2.00 (95% CI 0.72 to 3.28) months, compared to 12.0 (95% CI 2.40 to 21.6) months in the cGVHD group (p=0.084, Figure C); the median OS had no significant difference (Figure D). RD-alloCAR patients with or without cGVHD at PBMC collection did not differ in terms of the peak CAR T-cell expansion, CRS grades or OS.Conclusions: Compared with autoCAR, RD-alloCAR had similar efficacy but less severe CRS. For RD-alloCAR T-cell therapy, the presence of cGVHD at the time of PBMC collection was associated with fewer 6-month relapses. DD-alloCAR and RD-alloCAR T-cell therapies were effective and safe without causing significant GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

2027. Immune Correlates of Protection Against Herpes Zoster (HZ) in People Living with HIV (PLWH)

BackgroundHZ has high morbidity in immune-compromised hosts, including PLWH. Two vaccines against HZ are available, but their efficacy in PLWH is unknown. A surrogate marker of protection against HZ would facilitate efficacy studies of HZ vaccines in PLWH, as compared with HZ clinical outcome studies. To this goal, our study investigated the association of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) with incident HZ in PLWH.MethodsThe study used peripheral blood mononuclear cells (PBMC) cryopreserved at mean (SD) of 2.7 (2.5) months before HZ from 31 PLWH cases on ART with plasma vRNA < 200 c/mL, and CD4 counts ≥200 cells/µL. 31 non-HZ controls were matched to cases by CD4 count, age, sex, race, parent study, and ART duration. T cell subsets were measured by flow cytometry after ex vivo VZV- and mock-restimulation and in freshly thawed unstimulated PBMC using the following markers: CD3, CD8, CD25, CD28, CD39, CD57, CD127, CTLA4, FOXP3, IFNg, IL10, KLRG1, LAG3, PD1, perforin, TGFb, TIM3 and TNFa. Data were analyzed by paired t-tests.ResultsAt PBMC collection, PLWH had mean (SD) age 41 (10) years; 16 females; 28 White and 22 Black non-Hispanics; mean (SD) CD4 counts 501 (222) cells/µL; 56 (90%) vRNA < 50 c/mL; median ART duration 50 weeks. In unstimulated PBMC, HZ cases had higher %FOXP3+CD25+(CD4+) Treg compared with controls (P = 0.08). Cases generally had lower responses to ex vivo VZV-restimulation (after subtraction of mock) compared with controls, including %perforin+(CD8+) CTL (P = 0.07) and %IL10+(CD4+) (P = 0.04) and %TGFb+(CD4+) Treg (P = 0.07). %FOXP3+CD25+(CD4+) Treg in unstimulated PBMC did not correlate with VZV-specific %perforin+(CD8+) or %IFNg+(CD4+) effector T cells, but the VZV-specific effector T cell percentages correlated with each other (r = 0.24, P = 0.05).ConclusionAlthough limited by small numbers, this study showed that before development of HZ, PLWH had marginally increased number of Treg in blood and decreased VZV-specific responses. VZV-specific Treg and CTL were equally lower in cases compared with controls, suggesting that a general low VZV-specific T cell responsiveness precedes HZ in PLWH. The lack of association between circulating Treg and VZV-specific T cell responses suggests independent mechanisms of action.Disclosures All authors: No reported disclosures.

26 - Phase 1 Study of Elotuzumab in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Background: Elotuzumab is a humanized monoclonal antibody directed against SLAMF7 that is approved for use in relapsed multiple myeloma patients in combination with lenalidomide and dexamethasone. We hypothesized that the addition of elotuzumab and autologous peripheral blood mononuclear cell (PBMC) reconstitution to standard-of-care autologous hematopoietic stem cell transplantation (ASCT) and lenalidomide maintenance for consolidation therapy in myeloma patients will be safe and feasible. Methods: This is a Phase 1b, open-label, trial investigating safety of administration of elotuzumab and autologous PBMC in conjuction with high-dose melphalan and stem cell transplantation followed by lenalidomide maintenance. The primary objective of this study is to assess the safety and tolerability. The secondary objectives are to assess myeloma disease status and progression-free survival (PFS) after one year of treatment. The treatment plan is as follows: In addition to standard peripheral blood stem cell mobilization and harvest, subjects undergo steady-state leukopheresis for PBMC collection. Subjects receive standard melphalan conditioning (day −1) and autologous stem cell rescue (day 0). Autologous PBMC are reinfused on day +3 post-stem cell infusion and cycle 1 of elotuzumab 20 mg/kg IV is given on day +4. Subjects receive subsequent cycles of elotuzumab every 28 days up to cycle 12. Lenalidomide maintenance at 10 mg orally daily days 1-21 of every 28-day cycle begins with cycle 4 of elotuzumab, and may continue off study beyond cycle 12 at the investigator's discretion. Results: The study completed accrual of the planned 15 subjects. All 15 subjects are included in the safety population, completed the treatment protocol or came off study, and are evaluable. The majority of adverse events, including infusion reactions attributable to elotuzumab, were grade 2 or lower. Grade 3 or higher hematologic AEs, including anemia, neutropenia, lymphopenia, thrombocytopenia, and non-hematologic AEs including nausea, vomiting, and dehydration, were attributable to the ASCT procedure. Two subjects came off study before study completion; one for personal choice (VGPR at time of study withdrawal) and one for early progression. Thirteen subjects completed the study protocol. At the one-year post-ASCT time point, subjects were re-staged and the results are as follows: 1 PR, 6 VGPR, and 7 sCR. ORR was 93% (14/15) and one-year PFS was 93% (14/15). Correlative studies including tumor microenvironment immunophenotyping, MRD assessment, and T cell receptor sequencing are ongoing. These results will be available for presentation.

Phase 1 Study of Elotuzumab in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Introduction: Elotuzumab is a humanized monoclonal antibody directed against SLAMF7 that is approved for use in relapsed multiple myeloma patients in combination with lenalidomide and dexamethasone. This agent appears to have several modes of action, including facilitation of antibody-dependent, cell-mediated cytotoxicity (ADCC) through binding to SLAMF7 on myeloma cells and activation of natural killer (NK) cells to kill tumor cells through ligation of the target. We initiated a single-center, open label, phase 1 trial based on the hypothesis that the addition of elotuzumab and autologous peripheral blood mononuclear cell (PBMC) reconstitution to standard-of-care autologous hematopoietic stem cell transplantation (auto-SCT) and lenalidomide maintenance for consolidation therapy in myeloma patients after induction therapy will be safe and feasible. We hypothesize that early PBMC reconstitution post-auto-SCT will restore a viable NK cell population for activation by elotuzumab, which may target residual myeloma cells and promote tumor-specific humoral and cellular immune responses against myeloma cells. Subsequent maintenance therapy with elotuzumab and lenalidomide may amplify this response, resulting in long-term maintenance of the minimal residual disease state.Methods. This is a Phase 1b, open-label, trial investigating elotuzumab and autologous PBMC reconstitution with auto-SCT consolidation therapy and lenalidomide maintenance. The primary objective of this study is to assess the safety and tolerability of elotuzumab and autologous PBMC reconstitution in the setting of auto-SCT and lenalidomide maintenance in multiple myeloma patients. The secondary objectives are to assess myeloma disease status and progression-free survival (PFS) after one year of treatment. Subjects must achieve partial response or better by IMWG criteria with induction chemotherapy, be eligible for auto-SCT by institutional standards, and meet inclusion/exclusion criteria. Fifteen subjects are planned in this pilot study. The treatment plan is as follows: In addition to standard peripheral blood stem cell mobilization and harvest, subjects undergo steady-state leukopheresis for PBMC collection. Subjects receive standard melphalan conditioning (day -1) and autologous stem cell rescue (day 0). Autologous PBMC are reinfused on day +3 post-stem cell infusion and cycle 1 of elotuzumab 20 mg/kg IV is given on day +4. Subjects receive subsequent cycles of elotuzumab every 28 days up to cycle 12. Lenalidomide maintenance at 10 mg orally daily days 1-21 of every 28-day cycle begins with cycle 4 of elotuzumab, and may continue off study beyond cycle 12 at the investigator's discretion. Bone marrow aspirates and peripheral blood are collected for correlative studies at screening, cycle 2, cycle 4, and at the end of study after cycle 12. For the primary endpoint analysis, the safety population includes all subjects who received at least one dose of study treatment. The evaluable population constitutes all subjects who received at least four of the first five planned doses of elotuzumab.Results: Fourteen of the planned 15 subjects have been enrolled in the study. Demographic and staging data reflect the general transplant-eligible myeloma patient population at our institution. All 14 of these subjects are included in the safety population, having received at least 1 dose of elotuzumab. Nine of 14 subjects have completed at least 4 of the first 5 planned elotuzumab infusions and are evaluable. The majority of adverse events, including infusion reactions attributable to elotuzumab, have been grade 2 or lower. Grade 3 or higher hematologic AEs, including anemia, neutropenia, lymphopenia, thrombocytopenia, and non-hematologic AEs including nausea, vomiting, and dehydration, were attributable to the auto-SCT procedure. There were no delays in hematopoietic reconstitution observed. One episode of grade 3 hypertension was attributed to elotuzumab infusion and resolved with supportive care. No AEs were attributed to PBMC reconstitution.Conclusions: The combination of elotuzumab and PBMC reconstitution with standard auto-SCT and lenalidomide maintenance for consolidation therapy of multiple myeloma appears to be safe and feasible. One subject withdrew for personal reasons. The trial is ongoing and is expected to complete accrual and the clinical results will be updated for presentation. DisclosuresChari:Celgene: Consultancy, Research Funding; Array Biopharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Takeda: Consultancy, Research Funding. Geerlof:Bristol-Myers Squibb: Employment. Jagannath:Novartis: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Merck: Consultancy. Cho:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus, Inc.: Research Funding; Genentech Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Ludwig Institute for Cancer Research: Membership on an entity's Board of Directors or advisory committees.

Genome-Wide Changes in Peripheral Gene Expression following Sports-Related Concussion.

We conducted a prospective study to identify genome-wide changes in peripheral gene expression before and after sports-related concussion (SRC). A total of 253 collegiate contact athletes underwent collection of peripheral blood mononuclear cells (PBMCs) before the sport season (baseline). Sixteen athletes who subsequently developed an SRC, along with 16 non-concussed teammate controls, underwent repeat collection of PBMCs within 6 h of injury (acutely). Concussed athletes underwent additional sample collection at 7 days post-injury (sub-acutely). Messenger RNA (mRNA) expression at baseline was compared with mRNA expression acutely and sub-acutely post-SRC. To estimate the contribution of physical exertion to gene changes, baseline samples from athletes who subsequently developed an SRC were compared with samples from uninjured teammate controls collected at the acute time-point. Clinical outcome was determined by changes in post-concussive symptoms, postural stability, and cognition from baseline to the sub-acute time-point. SRC athletes had significant changes in mRNA expression at both the acute and sub-acute time-points. There were no significant expression changes among controls. Acute transcriptional changes centered on interleukins 6 and 12, toll-like receptor 4, and NF-κB. Sub-acute gene expression changes centered on NF-κB, follicle stimulating hormone, chorionic gonadotropin, and protein kinase catalytic subunit. All SRC athletes were clinically back to baseline by Day 7. In conclusion, acute post-SRC transcriptional changes reflect regulation of the innate immune response and the transition to adaptive immunity. By 7 days, transcriptional activity is centered on regulating the hypothalamic-pituitary-adrenal axis. Future efforts to compare expressional changes in fully recovered athletes with those who do not recover from SRC could suggest putative targets for therapeutic intervention.

Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient.

BackgroundHigh quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.ResultsThe mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 106 cells/ml, 93.3 %) were compatible to those obtained with Ficoll (1.34 × 106 cells/ml, 97.2 %). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.ConclusionsOur findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells’ handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0113-0) contains supplementary material, which is available to authorized users.

Abstract 2763: Utility of clinical biomarkers for detecting protein kinase CK2 inhibition: A report from the phase I trial of CX-4945

Abstract Introduction: CX-4945 is a first-in-class, orally administered small molecule that potently and selectively inhibits protein kinase CK2, a previously unexploited molecular target with well documented roles in many cancers. In vitro, CX-4945 selectively kills cancer cells by modulating key survival pathways, resulting in cell cycle arrest, inhibition of cell proliferation, and promotion of apoptosis. CK2 hyperactivation of the PI3K/Akt pathway promotes the phosphorylation of several proteins which we have exploited as biomarkers for CX-4945 activity. A validated laser scanning cytometry method has been developed to quantify the phosphorylation of p21 and Akt in cells, and this has been employed to characterize these substrates in circulating blood cells collected from patients in the phase I clinical trial of CX-4945. Procedures: Eligible patients with advanced solid tumors with progressive disease, or for whom there are no available standard therapies, were administered CX-4945 in successive dose cohorts using a standard 3×3 design at: 90, 160, 300, 460, and 700 mg per dose. Oral doses were administered twice daily for twenty-one consecutive days of a four week cycle. Serial plasma samples were collected for pharmacokinetic analysis on the first and final dosing days of Cycle 1 (i.e., Day 1 and Day 21). In addition, whole blood samples were collected at pre-treatment, 4 hours and 8 hours following the first dose of CX-4945 on Day 1 and Day 21, and peripheral blood mononuclear cells (PBMCs) were isolated for pharmacodynamic biomarker evaluations (specifically, total and phosphorylated forms of p21 and Akt). Plasma samples were also collected at these time points for quantification of plasma IL-6. Results: Seventeen patients with advanced solid tumors from five separate dose cohorts have received oral doses of CX-4945 to date, and all patients in the study participated in the collection of PBMCs. Biomarkers from patients in Cohorts 3 and 4 demonstrated changes in their profile consistent with the inhibition of CK2. CX-4945 displays general linearity in PK parameters between the dose cohorts, with a terminal half life of approximately 25 hours at steady state. Conclusions: To date, no DLTs have yet been observed, and the MTD remains to be defined in this Phase I study. Early signals suggest a modulation of the biomarker profile consistent with CK2 inhibition. Patient enrollment will continue, to further characterize CX-4945's safety, tolerance, pharmacokinetics and pharmacodynamic effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2763.

Gene expression profiling of peripheral mononuclear cells in lame dairy cows with foot lesions

Lameness is a major health issue and likely the single most common cause of pain and discomfort in dairy cattle. Appropriate treatment is delayed or neglected due, in part, to lack of reliable detection. Assessment of cows with lameness is currently limited to subjective visual scoring systems based on locomotion and posture abnormalities. These systems are unreliable to detect lameness, and therefore, a large number of cows remain undiagnosed. The objective of this research was to search for potential biomarkers for lameness-associated painful inflammatory foot lesions in dairy cattle using microarray-based gene expression profiling of peripheral blood mononuclear cells (PBMC). BOTL5 microarrays spotted in duplicate with cDNA representing bovine immune response genes were interrogated with cDNA samples in an eight-array, balanced complete block design with dye swap. Samples from eight lame cows with inflammatory foot lesions and from eight sound cows were pair-matched by age, weight, days in lactation, and pregnancy status at time of PBMC collection and directly compared with each other on individual arrays. Statistical analysis of resulting fluorescence intensity data revealed 31 genes that were putatively differentially expressed in lame versus sound cows ( P < 0.05). Of these, BLASTn analysis and gene ontology information showed that 28 genes had high similarity or homology to known human and/or rodent genes. Validation of 15 of these genes known to be important in inflammation and pain was carried out using relative quantitative real-time RT-PCR, which confirmed the up-regulation of interleukin (IL)-2 (12.68 ± 1.47-fold increase) and IL-10 (2.39 ± 0.55-fold increase), matrix metalloproteinase-13 (MMP-13) (10.44 ± 1.14-fold increase), and chemokine C–C motif receptor-5 (CCR5) (5.26 ± 1.05-fold increase), in lame relative to sound cows ( P ≤ 0.05). Similarly, granulocyte-macrophage colony-stimulating factor receptor alpha chain precursor (GM-CSF-R-alpha) (2.30 ± 0.63-fold increase) and IL-4 (2.06 ± 0.59-fold increase) showed a tendency ( P = 0.10) for up-regulation in lame compared to sound cows. PBMC co-expression of IL-2, MMP-13, CCR5 and IL-10, and potentially IL-4 and GM-CSF-R-alpha appears to be a promising, objective sign of lameness-related inflammatory foot lesions in dairy cattle. In conclusion, this study revealed potential biomarkers of the presence of foot lesions that could boost diagnostic accuracy of lameness and, ultimately, help identify animals in need of pain relief.

A randomized phase II study of Xcellerated T Cells with or without prior fludarabine therapy in patients with multiple myeloma (MM)

2582 Background: T cells from MM pts can be activated & expanded ex vivo using the Xcellerate Process, in which peripheral blood mononuclear cells (PBMC) are incubated with anti-CD3 & anti-CD28 antibody-coated magnetic beads (Xcyte Dynabeads). In a previous study (Siegel et al., ASH 2004), Xcellerated T Cells (XTC) administered to MM pts following autologous transplant led to accelerated lymphocyte recovery & clinical responses in the majority of pts. In the current study, pts with relapsed/refractory MM were randomized to XTC ± 1 cycle of fludarabine prior to XTC. Fludarabine was used to assess the influence of lymphoablation on T cell therapy; it has been reported to have no significant anti-tumor activity in MM (Kraut et al., Invest. New Drug, 1990). Methods: Pts underwent leukapheresis for collection of PBMC for the Xcellerate Process. All pts received a single dose of 60–100 x 109 XTC. Pts on the fludarabine arm received a single cycle (5 days at 25 mg/m2/day), completed 4 days prior to T cell infusion. Results: Of 30 planned pts, 28 have been enrolled, 19 treated & 16 are evaluable to date. 7 pts had received prior transplant. The median # infused cells was 77.1 x 109 (range 57.8–96.4 x 109), which were 98.9% CD3+. No serious adverse events related to XTC were reported. In the fludarabine arm (n=10), lymphocytes decreased from 1,212 ± 222/mm3 (mean ± SEM) to 444 ± 147 following fludarabine, & then increased to 1,840 ± 216 on Day 14 following T cell infusion. In the non-fludarabine arm (n=7), lymphocyte counts increased from 1,204 ± 148 to 3,218 ± 404 on Day 14. NK cells increased from 64 ± 17 to 104 ± 18 on Day 14 (n=17). On the fludarabine arm, 4 pts developed Grade 3 or 4 neutropenia & 2 developed Grade 3 or 4 thrombocytopenia. The fludarabine schedule was therefore decreased from 5 to 3 days. No significant decreases in M-protein have been observed to date. Conclusions: XTC were well-tolerated & led to increases in T lymphocytes. Fludarabine was lymphoablative, as anticipated, but caused neutropenia & thrombocytopenia. Additional follow-up & pts will be required to determine the activity of XTC in relapsed myeloma. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Xcyte Therapies Xcyte Therapies Xcyte Therapies

The effect of G-CSF on lymphocyte subsets and CD34+ cells in allogeneic stem cell transplantation

The effect of granulocyte colony-stimulating factor (G-CSF) on peripheral blood lymphocytes (PBL) and CD34+ cell frequency in the apheresis product has been determined in 25 healthy stem cell donors. Peripheral blood mononuclear cells (PBMNC) were collected after five days of G-CSF 10 μg/kg/day s.c., which was well tolerated. The median number of leukocytes increased eight-fold over that of pretreatment levels. Collection of PBMNC lasted a median of two (range, 1–3) days. The mean mononuclear cell (MNC) count and total lymphocyte percentage were 6.69×10 8/kg and 59.08%, respectively, and the frequency of CD34+ cell expression was 2.1% in the apheresis product. The frequency of CD3+, CD4+, CD25+, NK and CD122+ cell expressions in mobilized PBMNC and PBL showed no significant difference. However, the frequency of CD8+, CD8+28+, CD3+DR+, CD19+, CD20+ and CD22+ B cells expression in the apheresis product increased significantly compared to steady-state PBL. In contrast, the frequency of the CD11a+ and CD8+38+ cell expressions in the apheresis product was decreased compared to the steady-state PBL. The mean yield of CD34+ and CD3+ cells were 13.6×10 6 and 2.69×10 8/kg of recipient body weight (RBW), respectively. Following allograft all patients engrafted with >0.5×10 9/l neutrophil and ⩾20×10 9/l platelets on a median of day 13 and 12, respectively. Nine patients had grade II–IV acute GVHD and chronic GVHD occurred in eight patients. Four patients died due to transplant-related complications. There was one late engraftment failure which occurred on the fifth month. Thirteen patients are still alive. In conclusion, these results indicate that administration of G-CSF at 10 μg/kg/day in normal donors alters the lymphocyte subsets and there are significant differences in the lymphocyte contents of the recipients before apheresis and in apheresis product.

Use of Epstein-Barr virus-transformed, autologous B-lymphoblastoid cells as antigen-presenting cells for establishment and maintenance of dengue virus-specific, human cytotoxic T lymphocyte clones

We have been maintaining dengue virus specific CD8+ cytoxic T lymphocyte (CTL) clones by repeated stimulation using autologous peripheral blood mononuclear cells (PBMC) as antigen presenting cells (APCs). In the present study, Epstein-Barr virus (EBV)-transformed autologous lymphoblastoid cell lines (LCL) were compared with autologous PBMC as APCs for long term culture of a dengue virus-specific, HLA class I-restricted CD8+ CTL clone CB2.8. We substituted autologous LCL for autologous PBMC and maintained CB2.8 for several months. CB2.8 cultured using LCL as APCs maintained antigen specific cytolytic activity. No demonstrable difference in the specificity or in the level of cytolytic activity against a panel of target cells was noted between the CB2.8 maintained with LCL and those maintained with PBMC. Lysis of the target cells was blocked by the anti-HLA-class I antibody indicating that HLA class I-restriction was also maintained. We then compared autologous LCL with autologous PBMC in the establishment of CD4+ CTL clones from the PBMC of a dengue-1 immune donor. Dengue 1-specific clones were derived from limiting dilution cultures using either type of APCs. Similar numbers of dengue virus-specific CD4+ CTL clones were established using LCL or PBMC as APCs. These results indicate that autologous LCL act as APCs for long term culture of virus-specific CTL clones and represent a cost effective alternative to repeated collection of PBMC.