Mononuclear Cell Collection Research Articles

Development of a Uniform Apheresis Case Report Form forStandardized Collection of Apheresis Data.

Apheresis is performed worldwide for an increasing number of indications. The development of common data elements (CDE) for apheresis related areas may facilitate conduct of new research, enhance quality initiatives including benchmarking, and improve patient care. This report describes the systematic development of the Uniform Apheresis Case Report Form (UACRF) as part of the Apheresis in the United States (ApheresUS) program. A consensus panel of 17 diverse experts in apheresis, related specialties, and electronic case report form (eCRF), and database development was assembled. The panel met via online conferencing from November 17, 2020 to December 1, 2021. A draft document was posted online for public comment from October 11, 2021 to November 10, 2021. Feedback was collected using an online survey tool. The consensus panel revised the UACRF. This version was converted to an eCRF with additional changes made to improve usability in this format. The final version of the UACRF was created on August 24, 2023. The UACRF contains 16 modules: procedure and subject eligibility, patient demographics, general procedure information, laboratory parameters, vascular access, common procedure elements, eight procedure specific modules (mononuclear cell collection and seven therapeutic modalities), outcomes, and site information. A total of 137 data elements were created, including 57 with one or more subelements. The UACRF is the first systematic attempt to develop CDE for therapeutic apheresis and white blood cell collections. Further validation of the UACRF is necessary to confirm the tool's ability to collect the relevant data elements and determine the usability of the form.

Cell throughput and performance ratio as quality indicators on hematopoietic stem cell apheresis: A single-center experience

BackgroundBenchmarking in CD34+ cell apheresis is crucial for optimizing resources, ensuring consistent collection performance, and ultimately, decision-making algorithms to improve donor safety. Key performance indicators such as the “performance ratio” (PR) are applied routinely in some apheresis centers, whereas this report identifies the “cell throughput” (CT) as another quality indicator in apheresis. Material and methodsThis single-center study includes retrospective data from 117 aphereses. CT and PR were calculated based on the mononuclear cell collection (MNC) or continuous mononuclear cell collection (cMNC) protocols of the Spectra Optia® apheresis system, types of venous access, transplant settings, andmobilization regimens. ResultsCTs (× 106 CD34+ cells/min) were found to be greater in cMNC compared to MNC protocols (1.4 vs. 1.0, p = 0.0037), in allogeneic versus autologous (1.3 vs. 1.1, p = 0.0274), and in the mobilization regimen of G-CSF alone versus the G-CSF combined (1.3 vs. 1.0, p = 0.0249). In contrast, PR (%) was only statistically significant in favor of the cMNC protocol (213.0 vs. 186.8 for MNC). ConclusionsCT and PR are feasible quality indicators on CD34+ cell apheresis, are easy to calculate and implement, and have clinical and administrative implications. Analyzing CT and PR may strengthen the institutional criteria for selecting cMNC or MNC protocols; they may also be used to evaluate the performance of new personnel or cell separator devices or, eventually, trigger investigations for those aphereses under-collected by specific thresholds.

Prediction of CD34+ hematopoietic stem cells in healthy allogeneic stem cell donors on the Optia cell separator based on CE2: Which formula is more correlated with actual CD34+?

Peripheral blood-derived hematopoietic stem cells (HSCs) are widely used for various adult stem cell transplants. To obtain sufficient HSCs from healthy volunteer donors during the apheresis process and ensure that the donors are exposed to fewer apheresis-related side effects, calculation methods have been developed for the prediction of processed blood volume or CD34+ count. However, there is no consensus on a formula to predict the volume of blood to be processed or the number of stem cells to be obtained. This study aimed to estimate the predicted blood volume and CD34+ cell counts using collection efficiency (CE)-based formulas and evaluate their accuracy compared to the actual CD34+ cell counts. It also seeks to identify the factors that affect CE. Data from 397 healthy, unrelated stem cell donors were retrospectively analyzed. An algorithm using four different CE2 metrics (1st quartile, mean, 3rd quartile, and median) was developed to predict the volume of blood to be processed using the Spectra Optia continuous mononuclear cell collection procedure. When employing the mean CE2 algorithm, the results revealed a strong correlation (r = .894, p < .001) between predicted and actual CD34+ values. The study also identified strong associations between pre-apheresis CD34+, pre-apheresis leukocyte count, the use of two doses of G-CSF, and low CE2. These findings suggest that the mean CE2 algorithm could be a potent, straightforward, and accurate tool for predicting CD34+ stem cell counts in healthy allogeneic stem cell donors and potentially optimizing stem cell collection procedures.

Role of extracorporeal photopheresis in the management of acute and chronic graft versus disease: current status.

Extracorporeal photopheresis (ECP) is a therapy that combines the collection of mononuclear cells by apheresis, the addition of a photosensitizer (8-methoxisoralen), the illumination of the product with ultraviolet A light, and the immediate infusion of the product to the patient. Initially developed and approved to treat T-cell cutaneous lymphomas, soon started to be used to treat graft versus host disease (GvHD) developed after allogeneic hematopoietic-cell transplantation. The high response rate of ECP in skin, ocular, oral, pulmonary, and liver forms of chronic GvHD, the steroid-sparing effect, and the improved overall survival of treated patients, made ECP one of the second-line treatments used to treat steroid-resistant acute and chronic GVHD. Recently, the development of new drugs for treating GVHD has changed the position of ECP in the therapy of GVHD and has started to be used in combination with drugs for increasing the response rate to the treatment in severe or resistant forms of acute and chronic GVHD. ECP remains an essential therapeutic resource in the management of patients with refractory acute and chronic GVHD.

Pharmacokinetics and Safety of Remdesivir in Pregnant and Nonpregnant Women With COVID-19: Results From IMPAACT 2032.

Pregnant people with coronavirus disease 2019 (COVID-19) experience higher risk for severe disease and adverse pregnancy outcomes, but no pharmacokinetic (PK) data exist to support dosing of COVID-19 therapeutics during pregnancy. We report PK and safety data for intravenous remdesivir in pregnancy. IMPAACT 2032 was a phase 4 prospective, open-label, nonrandomized opportunistic study of hospitalized pregnant and nonpregnant women receiving intravenous remdesivir as part of clinical care. Intensive PK sampling was performed on infusion days 3, 4, or 5 with collection of plasma and peripheral blood mononuclear cells (PBMCs). Safety data were recorded from first infusion through 4 weeks after last infusion and at delivery. Geometric mean ratios (GMR) (90% confidence intervals [CI]) of PK parameters between pregnant and nonpregnant women were calculated. Fifty-three participants initiated remdesivir (25 pregnant; median gestational age, 27.6 weeks; interquartile range, 24.9-31.0 weeks). Plasma exposures of remdesivir, its 2 major metabolites (GS-704277 and GS-441524), and the free remdesivir fraction were similar between pregnant and nonpregnant participants. Concentrations of the active triphosphate (GS-443902) in PBMCs increased 2.04-fold (90% CI, 1.35-3.03) with each additional infusion in nonpregnant versus pregnant participants. Three adverse events in nonpregnant participants were related to treatment (1 grade 3; 2 grade 2 resulting in treatment discontinuation). There were no treatment-related adverse pregnancy outcomes or congenital anomalies detected. Plasma remdesivir PK parameters were comparable between pregnant and nonpregnant women, and no safety concerns were identified based on our limited data. These findings suggest no dose adjustments are indicated for intravenous remdesivir during pregnancy. NCT04582266.

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.

Handling the different requirements for commercial and investigational MNC collections by apheresis

BackgroundWith the success of chimeric antigen receptor T-cell (CAR-T) and similar cellular-based therapies, the demand for collection of autologous mononuclear cells by apheresis (MNC(A)) from blood by apheresis has increased. From an apheresis technical standpoint, the collection of MNC(A) is relatively straightforward, especially when compared with collection of hematopoietic progenitor cells (HPC(A)). Most of the collection for MNC(A) are performed for the commercial entities, who use the product for manufacturing cellular therapeutics. We have noticed discrepancies in the handling and apheresis processes required by different companies in obtaining essentially the same product (all companies in the study manufacture CAR-T-based products). We have analyzed the MNC collection requirements from all FDA-approved CAR-T cellular products and some investigational products collected at University of Nebraska Medical Center. We identified discrepancies in the process and suggested mitigation strategies. MethodsStep-by-step analysis of the collection requirements. Review of the current guidelines and recommendations on this issue. ResultsMultiple discrepancies in the collection process have been identified, even in the products collected for the same company. Practical approach of satisfying all the requirements based on University of Nebraska Medical Center experience has been suggested. ConclusionThe current recommendations from multiple sources were reviewed in discussion.

Collection efficiency of mononuclear cells in offline extracorporeal photopheresis: can processing time be shortened?

Extracorporeal photopheresis (ECP) is a well-established but lengthy and burdensome cell-based therapy for various diseases such as cutaneous T-cell lymphoma, graft-versus-host disease and organ rejection after transplantation. The number of mononuclear cells (MNCs) that needs to be collected to obtain a clinical response to ECP is still under debate. The purpose of this retrospective study was to determine the number of lymphocytes, monocytes and neutrophils in mononuclear cell products (MCP) by flow cytometry and the collection efficiency in the offline ECP setting. We collected data from 10 different patients undergoing 162 ECP procedures using the Spectra Optia device for MNC collection. White blood cell (WBC) count of MCP was determined using a hematology analyzer. MNCs were analyzed for CD45 and CD14 expression by flow cytometry to exactly determine the collected lymphocyte and monocyte fractions. Collected MCP showed high cell yields with 55.3×106/kg MNCs and 41.1×106/kg lymphocytes. MCP were characterized by high MNC (81.3%) and low neutrophils (18.7%) percentage. Mean collection efficiency for WBCs and for MNCs was 23.9% and 62.0%, respectively. The MNC fraction showed a moderate to high correlation between peripheral blood cell count of patients and MCP count. This study is one of a few reports showing the monocyte-to-lymphocyte relation in MCP for ECP determined by flow cytometry. In comparison to historical data from inline ECP, the offline ECP processing one total blood volume results in considerably higher cell yields. For this reason, and to reduce the burden on patients, we propose that the offline ECP processing time can be substantially reduced.

Collection efficiency and safety of large-volume leukapheresis for the manufacturing of tisagenlecleucel.

In patients with relapsed or refractory B cell acute lymphoblastic leukemia or B cell non-Hodgkin lymphoma (r/r B-ALL/B-NHL) with low CD3+ cells in the peripheral blood (PB), sufficient CD3+ cell yield in a single day may not be obtained with normal-volume leukapheresis (NVL). Large-volume leukapheresis (LVL) refers to the processing of more than three times the total blood volume (TBV) in a single session for PB apheresis; however, the efficiency and safety of LVL for manufacturing of tisagenlecleucel (tisa-cel) remain unclear. This study aimed to investigate the tolerability of LVL. We retrospectively collected data on LVL (≥3-fold TBV) and NVL (<3-fold TBV) performed for patients with r/r B-ALL/B-NHL in our institution during November 2019 and September 2023. All procedures were performed using a continuous mononuclear cell collection (cMNC) protocol with the Spectra Optia. Although pre-apheresis CD3+ cells in the PB were significantly lower in LVL procedures (900 vs. 348/μL, p < .01), all patients could obtain sufficient CD3+ cell yield in a single day with a comparably successful rate of final products (including out-of-specification) between the two groups (97.2% vs. 100.0%, p = 1.00). The incidence and severity of citrate toxicity (no patients with grade ≥ 3) during procedures was not significantly different between the two groups (22.2% vs. 26.1%, p = .43) and no patient discontinued leukapheresis due to any complications. LVL procedures using Spectra Optia cMNC protocol was well tolerated and did not affect the manufacturing of tisa-cel.

The Italian registry of therapeutic apheresis in SISTRA: Year of activity 2022

Therapeutic apheresis refers to a group of extracorporeal blood processing procedures used in the treatment of a variety of systemic diseases. These complex procedures are burdened by adverse reactions related to both procedures and underlying medical conditions.Given the importance of centralizing the collection and the analysis of information on therapeutic apheresis, the Italian National Blood Center (NBC), at the request of the Italian Scientific Society of Hemapheresis and Cell Manipulation (SIdEM), implemented the Italian Registry of Therapeutic Apheresis (IRTA) including it in the Information System of Transfusion Services (SISTRA), coordinated by the NBC.In 2022, a total of 34,702 therapeutic apheresis procedures was carried out in 8,781 patients, including paediatric patients, with an average of 3.9 procedures per patient. The 2022 IRTA data indicate that the patient with hematological and/or neurological disorders mainly turns to the apheresis centers. These results confirm the IRTA data from years 2020 and 2021. In the hematological field, the apheresis centers supply hematopoietic stem cells collection for autologous transplantation as well as mononuclear cell collection for extracorporeal photopheresis. With regard to the neurological field, myasthenia, chronic inflammatory demyelinating polyneuropathy and Guillain-Barré syndrome along with other neurological pathologies related to immune disorders are the most treated.In conclusion, this manuscript presents 2022 activity data of IRTA providing institutions and scientific societies with a wide range of information including type and number of therapeutic procedures, adverse events and patients’ outcome.

Evaluating the Impact of Cryopreservation of PBMCs on CAR-T Therapy Efficacy and Safety in DLBCL Patients: An Informative Approach to Optimize Manufacturing Strategies

Background: Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a pivotal treatment modality for relapsed/refractory (R/R) B-cell malignancies. However, some patients are unable to receive CAR-T therapy due to challenges in obtaining sufficient lymphocytes through leukapheresis, often resulting from intensive prior treatments or impaired lymphocyte function after repeated tumor reductions under high tumor burden. This issue underscores the need to explore strategies such as advance collection and cryopreservation of peripheral blood mononuclear cells (PBMCs), which could provide enhanced flexibility in the pre-CAR-T treatment planning process and potentially widen the accessibility of CAR-T therapy. Aims: This retrospective study compares the safety, efficacy, and adverse reactions of anti-CD19 CAR-T cell therapy for Diffuse Large B-Cell Lymphoma (DLBCL) patients, using cryopreserved versus freshly harvested PBMCs. Methods: A total of 162 R/R DLBCL patients treated with an anti-CD19-CD3zeta-4-1BB CAR-T cell therapy from January 2019 to April 2023 were included in this study, excluding those who received concurrent autologous stem cell transplants. The median age was 52 (15-78) years old, and the majority (87.7%) patients had stage III-IV disease. With a median IPI score of 2 (range, 0-5), 71.0% (115/162) had extranodal lesions and 18.0% (29/162) had bulky disease &amp;gt; 7 cm. Among these, 136 patients received CAR-T cells derived from cryopreserved PBMCs, which were frozen with a controlled rate freezer and CryoSure-DEX40 cryoprotectant and stored in the liquid phase of liquid nitrogen. The remaining 26 patients received CAR-T cells that were directly cultured from freshly collected lymphocytes without any cryopreservation. For all patients, CAR-T cells were freshly infused post-manufacture. The study primarily explores the attainment of CAR-T cell infusion dose (achieving 2x10 6 cells/kg), in vivo expansion of CAR-T cells, and the persistence of CAR-T cells in the body. Secondary aims include assessing overall survival (OS), progression-free survival (PFS), 3-month complete response (CR) rate and objective response rate (ORR). The incidence of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and levels of cytokines were also recorded. In addition, baseline characteristics of both groups were taken into analysis, including factors such as Eastern Cooperative Oncology Group (ECOG) performance status, International Prognostic Index (IPI) score, age, gender, presence of bulky disease, and extranodal lesions. For statistical analysis, either chi-square test or Fisher's exact test was employed for categorical variables, whereas continuous variables were assessed using the rank-sum test. Survival curves between groups were compared using the log-rank test. All P-values were two-tailed. Results: Baseline characteristics between the cryopreserved and fresh groups showed no significant differences in age, gender, IPI score≥3, ECOG score≥3, and extranodal lesions (P all &amp;gt; .05), while bulky disease prevalence was higher in the cryopreserved group (20.6% vs. 3.85%, P = .049). Metrics of CAR-T cell expansion, including meeting the infusion dosage benchmark of 2x10 6 cells/kg (46.1% vs. 44.9%, P = .90), in vivo CAR-T cell persistence (median duration: 21 days for both groups, P= .48), and the peak proportion of CART19 cells in lymphocytes (median peak: 49.6% vs. 35.2%, P = .41), revealed no significant differences between the fresh and cryopreserved groups. Assessments of CAR-T therapy safety revealed no significant differences in the incidence of grade 3 or higher ICANS and CRS, or peak levels of cytokines including interferon gamma, IL-6, IL-10, soluble CD25, and TNFα (P all &amp;gt; .05) between the two groups. Clinical efficacy measurements (3-month CR and ORR, 1-year OS and PFS) were similar between the two groups, with no statistically significant differences in CR (46.2% vs. 45.5%), ORR (69.2% vs. 61.9%), 1-year OS (75.4% vs. 64.1%, Fig 1), and 1-year PFS (52.1% vs. 44.5%, Fig 2) (P all &amp;gt; .05). Conclusion: Our study affirms that cryopreservation of PBMCs has no significant influence on the safety and efficacy of anti-CD19 CAR-T therapy for patients with R/R DLBCL. These findings widen the therapeutic flexibility and accessibility of anti-CD19 CAR-T therapy for DLBCL patients. Key words: PBMC Cryopreservation, CAR-T, DLBCL

Phase 1/2 Study of Donor-Derived Anti-CD33 Chimeric Antigen Receptor Expressing T Cells (VCAR33) in Patients with Relapsed or Refractory Acute Myeloid Leukemia after Allogeneic Hematopoietic Cell Transplantation

Background and Significance: Although allogeneic hematopoietic cell transplantation (alloHCT) is a potentially curative therapy for patients with high-risk acute myeloid leukemia (AML), relapse occurs in 30% to 40% of alloHCT recipients and is associated with a 2-year post-relapse survival rate of less than 20%. Chimeric antigen receptor (CAR) T cells targeting tumor-associated antigens have demonstrated efficacy in relapsed/refractory (R/R) acute lymphoblastic leukemia and other hematologic malignancies, but there is limited clinical experience with CAR T cell therapies for AML. Current challenges for autologous AML CAR T cell therapy include the inability to harvest adequate numbers of functional, undifferentiated T cells as CAR T cell starting material. In addition, the potential presence of AML blasts in an autologous cell collection can interfere with T cell proliferation and function during the manufacturing process. To circumvent these impediments in patients with R/R AML after alloHCT, VCAR33 represents a CD33-directed CAR T cell product generated from lymphocytes from each patient's prior allogeneic stem cell donor. CAR T cell manufacture from HLA-matched donors will likely not only eliminate delays and failures in production due to lymphopenia but also improve T cell function by using healthy starting material. CD33 is a preferential target for AML CAR T cell therapy as it is expressed on the majority (&amp;gt;80%) of AML blasts and because prior clinical experience with gemtuzumab ozogamicin (GO; tradename: Mylotarg TM) has demonstrated the safety and efficacy of targeting CD33. Study Design and Methods: Study NCTxxxxxxxx* is a multi-center phase 1/2 study evaluating the safety and preliminary efficacy of VCAR33, donor-derived allogeneic CAR T cells targeting CD33 (Figure 1). The CAR construct used for the manufacture of VCAR33 contains a lintuzumab-derived binding domain and a CD28 co-stimulatory domain. Key inclusion criteria include adult patients with relapsed or measurable residual disease positive (MRD+) AML after undergoing alloHCT with an 8/8 HLA-matched related or unrelated donor. Patients who have undergone alloHCT with trem-cel, a CD33-deleted allogeneic stem cell product intended to facilitate the post-HCT treatment with CD33-directed therapies by reducing on-target off-tumor hematotoxicity, may also participate. Patients will be assigned to one of two study arms based on their AML disease burden: Arm A for morphologic disease with ≥ 5% blasts in the bone marrow, or Arm B for MRD+ defined as &amp;lt; 5% blasts in the bone marrow with ≥ 0.1% CD33+ leukemia cells by flow cytometry. The patient's prior stem cell donor undergoes apheresis for collection of mononuclear cells, which are used as starting material for VCAR33 manufacturing. Patients will receive lymphodepletion on days -5 to -2 with fludarabine (total 120 mg/m 2) and cyclophosphamide (total 1000 mg/m 2) followed by infusion of VCAR33 on Day 0. Each study arm will enroll and escalate independently according to a standard 3+3 trial design, starting at 1 x 10 6 CAR T cells/kg for Dose Level 1. The dose limiting toxicity monitoring period is 28 days, at which time patients will undergo evaluation of disease response. Secondary endpoints include incidence of cytokine release syndrome, graft-vs-host disease, and clinical response. Exploratory endpoints aim to correlate VCAR33 properties with clinical response by characterizing T cell phenotypes within the VCAR33 product and by examining VCAR33 expansion and persistence after infusion. Follow up on trial will continue for up to 2 years after infusion followed by long term monitoring for up to 15 years. Patients may undergo second alloHCT for consolidation of remission or for rescue of prolonged cytopenia due to on-target off-tumor effects of VCAR33. *NCT number pending. Clinical trial information submitted to CT.gov on July 19, 2023.

Evaluating the efficiency and safety of large-volume leukapheresis using the Spectra Optia continuous mononuclear cell collection protocol for peripheral blood stem cell collection from healthy donors: A retrospective study.

Large-volume leukapheresis (LVL) refers to processing of more than three volumes of blood in a single session for peripheral blood stem cell collection. Recently, continuous mononuclear cell collection (cMNC) protocol has been developed using the Spectra Optia system, which is a widely used apheresis device. LVL using the novel protocol has been investigated in patients. However, the efficiency and safety of LVL in healthy donors using this protocol has not been characterized. Therefore, this study aimed to evaluate the efficiency and tolerability of CD34+ collection of LVL with the cMNC protocol in healthy donors. We retrospectively collected data on LVL (>3 total blood volume) and normal-volume leukapheresis (NVL) performed in healthy donors between October 2019 and December 2021. All procedures were performed using the cMNC protocol. Although pre-apheresis CD34+ cell count was lesser in LVL (23.5 vs. 58.0/μL, p < .001), CD34+ collection efficiency was comparable between LVL and NVL (61.2% vs. 61.4%, p = .966). Platelet loss was significantly higher in LVL compared to NVL (38.0% vs. 29.4%, p < .001), with no correlation between attrition of platelet and processing blood volume. Moreover, the incidence of citrate toxicity during procedures was comparable between the two groups (31.6% vs. 21.4%, p = .322). All LVL procedures could be completed without any adverse events. Allogeneic LVL procedure using Spectra Optia cMNC protocol was well tolerated by the donors and resulted in efficient collection of CD34+ cells, which was comparable to that of NVL.

Integrated study of systemic and local airway transcriptomes in asthma reveals causal mediation of systemic effects by airway key drivers

BackgroundSystemic and local profiles have each been associated with asthma, but parsing causal relationships between system-wide and airway-specific processes can be challenging. We sought to investigate systemic and airway processes in asthma and their causal relationships.MethodsThree hundred forty-one participants with persistent asthma and non-asthmatic controls were recruited and underwent peripheral blood mononuclear cell (PBMC) collection and nasal brushing. Transcriptome-wide RNA sequencing of the PBMC and nasal samples and a series of analyses were then performed using a discovery and independent test set approach at each step to ensure rigor. Analytic steps included differential expression analyses, coexpression and probabilistic causal (Bayesian) network constructions, key driver analyses, and causal mediation models.ResultsAmong the 341 participants, the median age was 13 years (IQR = 10–16), 164 (48%) were female, and 200 (58.7%) had persistent asthma with mean Asthma Control Test (ACT) score 16.6 (SD = 4.2). PBMC genes associated with asthma were enriched in co-expression modules for NK cell-mediated cytotoxicity (fold enrichment = 4.5, FDR = 6.47 × 10−32) and interleukin production (fold enrichment = 2.0, FDR = 1.01 × 10−15). Probabilistic causal network and key driver analyses identified NK cell granule protein (NKG7, fold change = 22.7, FDR = 1.02 × 10−31) and perforin (PRF1, fold change = 14.9, FDR = 1.31 × 10−22) as key drivers predicted to causally regulate PBMC asthma modules. Nasal genes associated with asthma were enriched in the tricarboxylic acid (TCA) cycle module (fold enrichment = 7.5 FDR = 5.09 × 10−107), with network analyses identifying G3BP stress granule assembly factor 1 (G3BP1, fold change = 9.1 FDR = 2.77 × 10−5) and InaD-like protein (INADL, fold change = 5.3 FDR = 2.98 × 10−9) as nasal key drivers. Causal mediation analyses revealed that associations between PBMC key drivers and asthma are causally mediated by nasal key drivers (FDR = 0.0076 to 0.015).ConclusionsIntegrated study of the systemic and airway transcriptomes in a well-phenotyped asthma cohort identified causal key drivers of asthma among PBMC and nasal transcripts. Associations between PBMC key drivers and asthma are causally mediated by nasal key drivers.

Apheresis collection of mononuclear cells for chimeric-antigen receptor therapies.

Collections of lymphocytes to be genetically modified to treat hematologic malignancies have seen a dramatic increase over the last few years as commercial products have been approved. Reports of new products in development that can possibly treat solid organ malignancies represent a massive change in the field. Apheresis is at the center of the collection of cells for the manufacture of these chimeric-antigen receptor therapy products. The expansion of these collections represents one of the areas of apheresis procedures growth. This review will summarize concepts important to this type of collection and variables that need to be optimized to obtain desired cell yields while increasing patients' safety.

Low cross reactivity between wild type and deamidated AAV can lead to false negative results in immune monitoring T-cell assays.

During gene therapy trials, immune responses against adeno-associated virus (AAV) vectors are monitored by antibody assays that detect the humoral and T-cell mediated cellular responses to AAV vectors. T cell assays commonly utilize the collection of patients' peripheral blood mononuclear cells (PBMCs) and stimulation with AAV-derived overlapping peptides. We recently described that spontaneous deamidation coincides with T cell epitopes in AAV capsids and that spontaneous deamidation may enhance or decrease immunogenicity in some individuals. This raised the concern for false negative results of antibody detection and PBMC immune monitoring assays because these assays use wild-type (WT) AAV or WT peptides for T cell re-stimulation and these peptides may not re-activate T cells that were stimulated with deamidated AAV capsid. To investigate this concern, we modeled the scenario by expanding T cells with deamidated peptides and evaluated the cross-reactivity of expanded T cells to WT peptides. In the majority of samples, cells that were expanded with deamidated peptides and restimulated with WT peptide had significantly lowered IL-2 and IFN-γ production. Spiking the four deamidated peptides to the WT peptide pool used for re-stimulation, restored the signal and corrected the performance of the assay. We also evaluated the impact of deamidation on anti AAV binding antibodies and did not observe a major impact on seroprevalence detection of AAV9. These data indicate that a high level ofdeamidation in AAV therapy may result in underestimation or even failure todetect immune responses against WT peptides during cellular immune monitoring.

Identification of Shared Immune Cells and Immune-Related Co-Disease Genes in Chronic Heart Failure and Systemic Lupus Erythematosus Based on Transcriptome Sequencing.

The purpose was to identify shared immune cells and co-disease genes in chronic heart failure (HF) and systemic lupus erythematosus (SLE), as well as explore the potential mechanisms of action between HF and SLE. A collection of peripheral blood mononuclear cells (PBMCs) from ten patients with HF and SLE and ten normal controls (NC) was used for transcriptome sequencing. Differentially expressed genes (DEGs) analysis, enrichment analysis, immune infiltration analysis, weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) analysis, and machine learning were applied for the screening of shared immune cells and co-disease genes in HF and SLE. Gene expression analysis and correlation analysis were used to explore the potential mechanisms of co-disease genes and immune cells in HF and SLE. In this study, it was found that two immune cells, T cells CD4 naïve and Monocytes, displayed similar expression patterns in HF and SLE at the same time. By taking intersection of the above immune cell-associated genes with the DEGs common to both HF and SLE, four immune-associated co-disease genes, CCR7, RNASE2, RNASE3 and CXCL10, were finally identified. CCR7, as one of the four key genes, was significantly down-regulated in HF and SLE, while the rest three key genes were all significantly up-regulated in both diseases. T cells CD4 naïve and Monocytes were first revealed as possible shared immune cells of HF and SLE, and CCR7, RNASE2, RNASE3 and CXCL10 were identified as possible key genes common to HF and SLE as well as potential biomarkers or therapeutic targets for HF and SLE.

A nomogram prediction of citrate reaction during mononuclear cell collection in solid tumor patients.

Citrate reaction is one of the main adverse events in peripheral blood mononuclear cell (MNC) collection. The aim of this study was to elucidate the risk factors for citrate reaction in patients with advanced solid tumor collection and to construct a nomogram to predict the risk. One hundred forty-eight patients with advanced solid tumor who underwent peripheral blood MNC collection in our hospital between January 2021 to December 2021 were selected. The general data, creatinine level before collection, Ca2+ concentration before collection, absolute value of monocyte lymphocytes before collection, circulating blood volume, anticoagulant dosage, and blood collection duration were included in Logistic regression analysis to identify the risk factors of citrate reaction. According to the results of the multivariate logistic model, nomogram was established and receiver operating characteristic (ROC) curve was drawn to evaluate the predictive value of the model. Among the 148 solid tumor patients, 35 patients (23.6%) of the 148 patients developed citrate reaction. Multivariate analysis showed that the risk factors for citrate reaction in the process of collection included sex (odds ratio [OR]=6.718; 95% confidence interval [95% CI]: 2.191-20.594, P=.001), age (OR=0.957; 95% CI: 0.921-0.996, P=.03), and processed circulating blood volume (OR=1.001; 95% CI: 1.000-1.002, P=.01). Logistic regression can analyze independent risk factors and establish risk prediction model. The predictive performance of the model is good, and the area under ROC curve is 0.799. The MNC collection process is safe. The incidence of citrate reaction in the collection of peripheral blood MNCs from patients with advanced solid tumor is related to the age, gender, and processed circulating blood volume of patients. The nomogram can be used to assess a patient's risk of citrate reaction.

Impact of Mobilization Strategies on Peripheral Blood Stem Cell Collection Efficiency and Product Quality: A Retrospective Single-Center Study.

Autologous stem cell transplantation is routinely used in the management of several hematological diseases, solid tumors, and immune disorders. Peripheral blood stem cell (PBSC) collection performed by apheresis is the preferred source of stem cells. In this study, the potential impact of mobilization regimens on the performance of the Spectra Optia® continuous mononuclear cell collection system was evaluated. We performed a retrospective data analysis for patients undergoing autologous PBSC collection at the Medical University Vienna, Vienna General Hospital between September 2016 and June 2018. Collections were divided into two main groups according to the mobilization regimen received: without (210 collections) or with (99 collections) plerixafor. Assessed variables included product characteristics and collection efficiency (CE). Overall, product characteristics were similar between the groups. Median CD34+ CE2 was 50.1% versus 53.0%, and CE1 was 66.9% versus 69.9% following mobilization without and with plerixafor, respectively; the difference was not statistically significant. Simple linear regression showed a very weak positive correlation between the mobilization method and CE1 or CE2 (mobilization with plerixafor increased CE2 by 4.106%). In conclusion, the Spectra Optia® apheresis system led to high CE and a good quality of PBSC products when mobilization regimens with or without plerixafor were used.

Evaluation of cell collection efficiency in non-mobilized adult donors for autologous chimeric antigen receptor T-cell manufacturing.

Data about collection efficiency 1 (CE1), which takes into account blood cell counts before and after collection, thus providing a more accurate estimate, in the collection of autologous T lymphocytes by apheresis for chimeric antigen receptor (CAR) T-cells remain scarce. We evaluated donor- and procedure-related characteristics that might influence the CE1 of lymphocytes. We retrospectively reviewed all mononuclear cell (MNC) collections) performed for CAR T-cell manufacturing in our institution from May 2017 to June 2021 in adult patients. Age, gender, weight, total blood volume (TBV), prior haematopoietic cell transplant, diagnosis, days between last treatment and apheresis, pre-collection cell counts, duration of apheresis, TBV processed, vascular access, inlet flow and device type were analysed as potential factors affecting CE1 of lymphocytes. A total of 127 autologous MNC collections were performed on 118 patients diagnosed with acute lymphoblastic leukaemia (n=53, 45%), non-Hodgkin lymphoma (n=40, 34%), multiple myeloma (n=19, 16%), and chronic lymphocytic leukaemia (n=6, 5%). The median CE1 of lymphocytes was 47% (interquartile range: 32%-65%). In multiple regression analysis, Amicus device was associated with higher CE1 of lymphocytes (p=0.01) and lower CE1 of platelets (p < 0.01) when compared with Optia device. The knowledge of the MNC and lymphocyte CE1 of each apheresis device used to collect cells for CAR T therapy, together with the goal of the number of cells required, is essential to define the volume to be processed and to ensure the success of the collection.