Number Of Peripheral Blood Mononuclear Cells Research Articles

Disparity of gene expression in coronary artery disease: insights from MEIS1, HIRA, and Myocardin.

Coronary artery disease (CAD) in young adults can have devastating consequences. The cardiac developmental gene MEIS1 plays important roles in vascular networks and heart development. This gene effects on the regeneration capacity of the heart. Considering role of MEIS1 in cardiac tissue development and the progression of myocardial infarction this study investigated the expression levels of the MEIS1, HIRA, and Myocardin genes in premature CAD patients compared to healthy subjects and evaluated the relationships between these genes and possible inflammatory factors. The study conducted a case-control design involving 35 CAD patients and 35 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were collected, and gene expression analysis was performed using real-time PCR. Compared with control group, the number of PBMCs in the CAD group exhibited greater MEIS1 and HIRA gene expression, with fold changes of 2.45 and 3.6. The expression of MEIS1 exhibited a negative correlation with IL-10 (r= -0.312) expression and positive correlation with Interleukin (IL)-6 (r = 0.415) and tumor necrosis factor (TNF)-α (r = 0.534) gene expression. Moreover, there was an inverse correlation between the gene expression of HIRA and that of IL-10 (r= -0.326), and a positive correlation was revealed between the expression of this gene and that of the IL-6 (r = 0.453) and TNF-α (r = 0.572) genes. This research demonstrated a disparity in expression levels of MEIS1, HIRA, and Myocardin, between CAD and healthy subjects. The results showed that, MEIS1 and HIRA play significant roles in regulating the synthesis of proinflammatory cytokines, namely, TNF-α and IL-6.

Abstract 4: A novel humanized mouse model for study of T cells and NK cells in the tumor microenvironment

Abstract Humanized mouse models can be useful for in vivo preclinical evaluation of mechanisms underlying cancer immunotherapy. Most such models involve systemic engraftment of immunocompromised mice with human lymphoid and hematopoietic cells, often using normal donor peripheral blood mononuclear cells (PBMCs), followed by inoculation with human tumor. The ability to control and modify immune cells within the tumor microenvironment (TME), especially human NK cells, is limited in such models. Here, we report the development of a mouse model designed to control the number of human T cells and NK cells in the TME. 18 mice and pre-validated PBMCs were obtained from Charles River. Six groups of 3 mice each were inoculated with Raji lymphoma cells. Group 1 was inoculated with Raji cells only and received no PBMCs. Raji cells were mixed PBMCs for groups 2-4. Group 5 was systemically engrafted with PBMCs 12 days prior to Raji inoculation and Group 6 was systemically engrafted with PBMCs 10 days after Raji inoculation. All mice developed tumors with similar growth characteristics. Serial fine needle aspirates (FNAs) of the resulting tumors were evaluated by flow cytometry. Results of FNAs are summarized below. Tumors were harvested at the end of experiment, typically 27 days after tumor inoculation, and evaluated by immunohistochemistry. Both T cells and NK cells were evenly distributed within tumors at the time of harvest. We conclude co-injection of tumor and PBMCs results in engraftment of both T cells and NK cells within the TME for up to 27 days, with T cells and NK cells distributed evenly within the tumor. Mixing higher numbers of PBMCs with tumor cells resulted in increased numbers of T cells and NK cells in the TME throughout the experiment. Matrigel did not improve engraftment or persistence of T or NK cells in the TME. We conclude mixing PBMCs with Raji tumor cells prior to tumor inoculation into immunocompromised mice is an effective approach when a humanized model with human T cells and NK cells within the TME is desired. Group Description % T cells in tumor at 17 days % NK cells in tumor at 17 days 1 No PBMCs 0 0 2 PBMCs mixed with Raji at 10:1 ratio 8.6 1.5 3 PBMCs mixed with Raji at 2:1 ratio 20.3 2.7 4 PBMCs and Raji in matrigel 9.2 0.6 5 PBMCs systemically engrafted 12 days before Raji inoculation 23.2 0.1 6 PBMCs systemically engrafted 10 days after Raji inoculation 19.6 0.2 Citation Format: Jyoti Arora, Caitlin D. Lemke-Miltner, George J. Weiner. A novel humanized mouse model for study of T cells and NK cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4.

Abstract 18 Developing a Potency Assay for Cord Tissue MSCs

IntroductionThe potency of mesenchymal stromal cells (MSCs) can be assessed by their ability to suppress proliferation of third party stimulated peripheral blood T cells. This correlates with decreased cellular production of IL-2Ra.ObjectiveWe optimized a potency assay for human cord tissue MSCs (hCT-MSC) in clinical trials for subjects with neuroinflammation. Originally, this T cell suppression assay required radioactive tritiated thymidine, rendering the assay difficult to use in a quality control laboratory for product release. Accordingly, we are developing a modified assay that eliminates the tritiated thymidine and instead tests for production of IL-2Rα in harvested supernatants.MethodsThe modified assay utilizes freshly thawed hCT-MSCs plated in a 96-well Corning CellBIND plate in Fuji Prime-XV XSFM MSC expansion medium at three concentrations. The hCT-MSCs are incubated 1-6 days at 37°C with humidified 5% CO2 until 80%-100% confluent. At this point, three different, previously qualified, normal control peripheral blood mononuclear cells (PBMCs) are suspended in RPMI 1640 with HEPES medium and 10% fetal bovine serum (FBS) with and without Dynabead (Human T-Activator CD3/CD28). After 4 additional days in culture, 75 µL of supernatant is removed and frozen at –80°C for future assaying of IL-10, TNFα, IFNg, and IL-2Ra using the Ella (Bio-Techne). IL-2Ra was determined to have the best correlation with the original method of suppression of proliferation. In the original radioactivity method after 4 days, the cells were pulsed with tritiated thymidine, harvested 6-10 hours after pulsing, and counted in the MicroBeta2 counter.ResultsSeventeen hCT-MSC lines were then tested in both assays. The passing criteria was >70% suppression in the radioactivity assays or >70% decrease in IL-2Ra production in the Ella assay. All 17 hCT-MSC lots passed in the radioactivity-based assay, while 16/17 lines passed using the IL-2Rα assay. Subsequently, the number of PBMCs plated per well and the number of days the PBMCs were in culture were optimized to maximize production of IL-2Rα.DiscussionWe developed a potency assay for MSCs that eliminates the use of radioactive reagents and usable as a release assay in manufacturing of hCT-MSC.

A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs.

Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration.

Static Adhesion Assay for Human Peripheral Blood Mononuclear Cells.

Blood endothelial cells (ECs) constitute the primary physical barrier to be crossed by circulating leukocytes, once attracted to a site of ongoing inflammation/infection. Upon a pro-inflammatory stimulus, such as tumor necrosis factor (TNF), ECs upregulate adhesion molecule expression to favor the adhesion and, subsequently, the transendothelial migration of the attracted lymphocytes. To address the ability of a cell to transmigrate through a monolayer of ECs, the classical transmigration assay is usually performed (Muller and Luscinskas, 2008). In the present protocol, adapted from Safuan et al. (2012), we describe an in vitro assay for assessing the functionality of the second step of the transendothelial migration, i.e., the firm adhesion of peripheral blood mononuclear cells (PBMCs) to ECs, under static conditions. By pre-incubating primary human umbilical cord ECs (HUVECs) with either innate lymphoid cell progenitors (ILCPs) or TNF, we were able to upregulate adhesion molecules on the EC surface. Then, by adding total PBMCs, we were able to both quantitatively and qualitatively analyze the cellular subtype and number of PBMCs that adhered to the pre-treated ECs. The important advantage of this technique is the possibility to perform functional studies on ECs biology since, differently from transwell-based strategies, it allows a good recovery of ECs at the end of the assay. Overall, this assay enables to interrogate how/if different stimulations/cell types can influence EC ability to retain PBMCs in vitro, under static conditions. Graphic abstract: The workflow of the Static Adhesion Assay.

Abstract PR-001: Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma

Abstract Introduction: Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The C-X-C chemokine receptor type 4 (CXCR4)/chemokine (C-X-C motif) ligand 12 (CXCL12) axis is a key immunosuppressive pathway where CXCL12 released by cancer-associated fibroblasts (CAF) binds to its receptor CXCR4 expressed by cytotoxic CD8+ T cells (CTL) which results in their decreased abundance within tumors. Encouraging preclinical and clinical data from mouse PDAC models and human PDAC tumor sections have shown T cell tumor accumulation after co-inhibition of CXCR4 and PD1/PDL1, however number of tumor regressions were limited. Results: To further explore the mechanism by which inhibition of the CXCR4/CXCL12 pathway modulates the immune TME in triggering an anti-tumor immune response in human PDAC, we generated an autologous co-culture system with patient-derived tumor organoids (PDTO) and peripheral blood mononuclear cells (PBMC). PDAC tumor tissue from surgical resection was used to generate lines of PDTO that recapitulate the histological and genetic characteristics of the original tumor. We demonstrate that tumor-specific reactive T cells can be obtained in PDAC by co-culture with autologous PDTO, with up to 5% of CD8+ T cells producing IFNγ after tumor organoid stimulation. No T cell reactivity was observed against normal pancreatic organoids, and blocking tumor MHC I/MHC II decreased the proportion of activated T cells. Ex vivo migration assays were established to better understand the role of CXCL12 in PDAC tumor T cell exclusion. Tumor organoids attracted autologous PBMC and addition of recombinant CXCL12 decreased PBMC migration, while treatment of PBMC with the CXCR4 inhibitor AMD3100, rescued the number of PBMCs that were able to migrate towards the tumor organoids. PBMC infiltrated PDTO-containing but not empty Matrigel domes. Addition of AMD3100 increased PBMC infiltration within the Matrigel dome containing PDTOs. Finally, we found that pre-treatment of PBMC with AMD3100 increased T cell reactivity against PDTO, and that T cell activation was further increased when stimulated with PDTO pre-treated with chemotherapy. Conclusions: We found that CXCL12 modulates the migration of T cells towards tumor organoids generated from pancreatic cancer tissue, and that inhibition of CXCR4 not only increases the migration potential, but also increases tumor-specific T cell activation. The use of ex vivo autologous co-culture and migration assays provide a unique strategy to identify modulators that may enhance T cell mediated neoplastic cell death to guide therapeutic intervention in PDAC. Citation Format: Emily Alouani, Ilenia Pellicciotta, Winston Wong, Alexander S. Thomas, Michael D. Kluger, Anna M. Chiarella, Anil K. Rustgi, Gulam A. Manji. Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-001.

Planktonic and Biofilm-Associated Pseudomonas aeruginosa and Staphylococcus epidermidis Elicit Differential Human Peripheral Blood Cell Responses.

Despite the considerable progress made in recent years, our understanding of the human immune response to microbial biofilms is still poor. The aim of the present study was to compare the in vitro response of human peripheral blood mononuclear cells (PBMC) to biofilms and planktonic cells of Pseudomonas aeruginosa and Staphylococcus epidermidis, two bacterial species particularly relevant in patients with cystic fibrosis or undergoing endovascular catheterization, respectively. PBMC isolated from healthy donors were co-cultured with 24 h-old biofilms or with exponentially growing cells of both species. Following 24 h of co-culture, the expression of early activation markers and the levels of cytokines in the culture supernatants were assessed by flow cytometry, while biofilm biomass and architecture were evaluated by crystal violet staining, CFU count, and confocal microscopy. Around 20% of PBMC was activated in response to both biofilms and planktonic cells of P. aeruginosa. In contrast, planktonic cells of S. epidermidis induced a statistically higher degree of activation than their biofilm counterpart (25% versus 15%; p < 0.01). P. aeruginosa biofilms stimulated pro-inflammatory (TNF-α, IL-1β, IFN-γ, and IL-6) and anti-inflammatory (IL-10) cytokine production at statistically significant levels higher than its planktonic counterpart, while an opposite trend was observed with S. epidermidis. Differences in the architecture of the biofilms and in the number of PBMC infiltrating the biofilms between the two bacterial species may at least partially explain these findings. Collectively, the results obtained highlighted marked differences in the host–cell response depending on the species and the mode of growth (biofilms versus planktonic cultures), allowing speculations on the different strategies adopted by P. aeruginosa and S. epidermidis to persist in the host during the course of chronic infections.

Evaluation of immune responses in a novel ovarian cancer immune-oncology three-dimensional cell culture model

<h3>Objectives:</h3> We aimed to develop and validate a novel ovarian cancer 3D immune oncology co-culture model consisting of high grade ovarian cancer tumor spheroids cultured with immune cells. We evaluated outcomes including tumor spheroid viability, ability to visualize immune cell migration and invasion, cellular expression of immune factors, and cell death resulting from immune co-culture. <h3>Methods:</h3> Spheroids were grown in 96-well U-bottom microplates using SKOV3 and OVCAR8 cell lines. Spheroids were then co-cultured with human peripheral blood mononuclear cells (PBMCs) from gynecologic cancer patients in 96-well 5 µm Transwell plates to allow for immune cell migration and invasion into spheroids. Confocal microscopy of live, labeled cells was performed to visualize migration and spheroid invasion. Flow cytometry was performed to determine spheroid viability over time and expression of immune factors MHC1 and PD-L1 on spheroid cells. Cancer cell death resulting from immune cell co-culture was quantified by propidium iodide immunofluorescence and compared to cell death in spheroids in the absence of PBMC or in spheroids treated with carboplatin. <h3>Results:</h3> SKOV3 and OVCAR8 spheroids became highly compacted after 48-72 hours in culture and retained excellent viability over 1 week. Strong e-cadherin expression was visible in both cell types at 48 hours. Live imaging of spheroids co-cultured with PBMC revealed robust invasion of the spheroids, which increased with increasing PBMC numbers and with time. MHC1 and PD-L1 levels were measured on spheroids alone cultured over 72 hours. Robust expression of MHC1 was observed on SKOV3 cells but was almost absent on OVCAR8 cells. Conversely, prominent expression of PD-L1 was noted on OVCAR8 cells. Immune cell killing of cancer cells was increased compared to controls for both SKOV3 and OVCAR8. OVCAR8 cells demonstrated different dynamics of cell death than SKOV3, with cell death in OVCAR8 peaking at 72 hours and cell death in SKOV3 peaking at 48 hours. <h3>Conclusions:</h3> Our findings demonstrate feasibility and reproducibility of an ovarian cancer 3D immune oncology co-culture model. As compared to traditional 2D tissue culture, this new technique may offer a more physiologic representation of cancer, its microenvironment, and its interaction with the immune system, which may improve preclinical study of cancer and antineoplastic agents.

Longitudinal analysis of dynamic changes of T-lymphocytes during multimodal treatment of patients with inoperable stage III NSCLC.

e20503 Background: Acute lymphocytopenia is associated with poor survival in solid cancers treated with multimodal therapy. A prospective analysis of peripheral blood mononuclear cells (PBMCs) during multimodal treatment in inoperable stage III NSCLC patients was performed to assess a correlation of T-lymphocytes changes with 6-months progression-free survival rates (PFS6M). Methods: Twenty patients at median age of 65.5 years (range 33-77), 85% male, 55% with adenocarcinoma and 40% with squamous cell carcinoma, were prospectively enrolled in this study. Eighteen (90%) patients received platinum-based concurrent chemo-radiotherapy (cCRT); seven (35%) patients additional concurrent and/or sequential immune check-point inhibition (four patients nivolumab and three durvalumab); patients treated with nivolumab received induction chemotherapy. Thoracic irradiation (TRT) was applied in all patients with median cumulative dose in equivalent 2Gy fractions (EQD2) of 64Gy (range: 52-65Gy). Peripheral blood was collected 5-10 days before treatment begin (A1), on the last day of TRT (RTend), and during follow-up. Samples were analyzed using polychromatic flow cytometry. Results are reported for three time-points: A1, RTend, and 6 months after TRT (C3) or the last sample available before that time-point. Results: From A1 to RTend, 16 (80%) patients experienced severe T-cell (CD3+, CD3+CD4+, CD3+CD8+) depletion, including 3 (15%) patients who received two doses of concurrent nivolumab. T-lymphocyte nadir was independent of the absolute numbers of PBMCs before treatment begin. In two patients, T-cell count remained stable, and increased in two other patients. No correlation of dynamic changes from A1 to RTend with PFS6M was observed. From RTend to C3, T-lymphocytes recovered in 11 (55%) patients; in 6 (30%) T-cell count further decreased or remained at very low levels. For total CD3 T-cells, CD3+CD4+ and CD3+CD8+ subsets, progressive disease in the first six months after TRT was associated with a decrease of median values (P = 0.03 for total CD3+ and CD3+CD4+, P = 0.08 for CD3+CD8+ T-cells). In contrast, an increase of all medians was associated with PFS6M (P = 0.007 for total CD3+, P = 0.002 for CD3+CD4+, P = 0.06 for CD3+CD8+ T-cells). Conclusions: There is a significant difference between patients with regards to T-lymphocytes recovery after the end of TRT, which is predictive for PFS6M, with poor median recovery observed in patients with early progress.

Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells.

Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.

Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells

Human Vγ9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 μM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 17(3): 460-469 (2021) ©Biomedical Informatics (2021) 461 at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.

Phenotypical Changes of Hematopoietic Stem and Progenitor Cells in Sepsis Patients: Correlation With Immune Status?

Background: Sepsis is life-threatening organ dysfunction associated with high risk of death. The immune response of sepsis is complex and varies over time. The immune cells are derived from hematopoietic stem and progenitor cells (HSPCs) which can respond to many infections. Our previous study found that sepsis causes HSPC dysregulation in mouse. But few studies have previously investigated the kinetics of HSPC and its contribution to immune system in sepsis patients. Purpose: We aimed to identify the kinetics of HSPCs and their contribution to immune system in sepsis patients. Methods: We enrolled eight sepsis patients and five healthy control subjects. Peripheral blood (PB) samples from each patient were collected three times: on the first, fourth, and seventh days, once from each healthy control subject. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation and stained with cocktails of antibodies. Populations of HSPCs and their subpopulation were analyzed by flow cytometry. Immune cells were characterized by flow cytometry and blood cell analysis. Correlations between HSPCs and immune cells were analyzed using the Pearson correlation test. Results: We found that the frequency of HSPCs (CD34+ cells and CD34+CD38+ cells) in sepsis patients on day 4 was significantly higher than that in the healthy controls. The most pronounced change in subpopulation analysis is the frequency of common myeloid progenitors (CMPs; CD34+CD38+CD135+CD45RA−). But no difference in the immunophenotypically defined hematopoietic stem cells (HSCs; CD34+CD38−CD90+CD45RA−) in sepsis patients was observed due to rare HSC numbers in PB. The number of PBMCs and lymphocytes are decreased, whereas the white blood cell (WBC) and neutrophil counts were increased in sepsis patients. Importantly, we found a negative correlation between CD34+ on day 1 and WBC and lymphocytes on day 4 from correlation analysis in sepsis patients. Conclusion: The present study demonstrated that the HSPC and its subpopulation in sepsis patients expanded. Importantly, the changes in HSPCs at early time points in sepsis patients have negative correlations with later immune cells. Our results may provide a novel diagnostic indicator and a new therapeutic approach.

Abstract 4281: Overcoming preanalytical variabilities for peripheral blood mononuclear cells and exosome profiling in the IMMUcan multicentre study

Abstract IMMUcan is a joint public-private partnership to advance our understanding of how tumors and the immune system interact and the effect of therapeutic interventions. The project will collect tumor biopsies and peripheral blood from up to 3,000 patients across 13 European countries for multi-omic analyses. Here, we assessed the impact of sample collection and processing conditions on the quality of peripheral blood mononuclear cells (PBMCs). We also compared circulating exosomes obtained from plasma and serum to determine the ideal source for the study. We collected peripheral blood from 9 healthy donors across 3 centers in different countries. PBMC isolation and freezing were either performed locally at 3, 8, 24, 48 and 72 hours delays or alternatively shipped to a designated biobank for central processing at 8, 24, 48 and 72 hours delays. PBMCs were assessed for yield, viability and frequencies of immune cell populations by flow cytometry. Serum vs plasma derived exosomes were compared using nanoparticle tracking analysis for size distribution and immuno-dotblot for protein levels of HER1, HER2, HER3, cMET, P4HA1, S1000A9, PD-L1, and ALIX. Centralized processing yielded more consistent results with an overall improvement in PBMC numbers and viability. The delay between blood withdrawal and PBMC isolation had a noticeable impact on PBMC quality when it exceeded 24 hours. Between 24 and 48 hours, we observed a significant and donor dependent decrease in viability and a major increase in granulocytic contamination. Relative frequencies of NK cells were the most sensitive to processing delays during the first 24 hours. Despite a slight global decrease in viability at 24 hours, the relative proportions of other major immune subsets remained stable. Regarding exosomes, plasma-exosomes had a significantly higher modal size compared to serum-derived. Particle abundance was minimally higher in the serum-exosomes. There were no significant differences in the levels of most proteins except for HER3 and c-MET, which were significantly upregulated in plasma exosomes. We opted to centralize PBMC processing to one biobank to minimize variability. As it was logistically impossible to ensure shipments and processing within 24 hours for all recruitment sites, we limited sample inclusion up to 48 hours. This will be accounted for by tracking the number of granulocytes in future CyTOF measurements and including the delay durations in subsequent multi-variate analyses. Exosomes from serum and plasma will be included in the study as the data showed that both sources produced exosomes of adequate quality yet were different enough in protein levels to merit further investigation. Funding: IMI2 JU grant agreement 821558, supported by EU's Horizon 2020 and EFPIA. Citation Format: Rami Mustapha, Valentina Pomella, Jose Vicencio, Silvia Lopez-Lastra, Christelle Bahlawane, Wim Ammerlaan, Henoch S. Hong, Marie Morfouace, Caoimhe Vallely-Gilroy, Vassili Soumelis, Emanuela Romano, Sabine Tejpar, Tony Ng, Fay Betsou, IMMUcan consortium. Overcoming preanalytical variabilities for peripheral blood mononuclear cells and exosome profiling in the IMMUcan multicentre study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4281.

A profile of circulating vascular progenitor cells in human neovascular age-related macular degeneration

Background/objectiveA subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects.Materials and methodsBlood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion, the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR.ResultsThere was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media, and markers such as NG2 and αSMA in mesenchymal media. However, analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population.ConclusionsCD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However, nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease.

Cellular therapy: Adoptive immunotherapy with expanded natural killer cells

Adoptive immunotherapy with natural killer cells was pioneered 30years ago in human clinical trials with the development of cytokine-induced killer cells-unfractionated peripheral blood mononuclear cell (PBMC) populations activated overnight with IL-2. Higher doses were subsequently made possible through the advent of steady-state apheresis, allowing the collection of PBMC numbers equivalent to an entire adult blood volume, and increased purity made feasible through magnetic CD3-depletion and/or CD56-selection methods. Still, these approaches rarely achieved clinical dosing above a single infusion of 108 NK cells/kg, except with substantial donor-recipient size mismatch (eg, parents donating cells to children). To address this shortcoming, leukemia cell lines with NK cell-like function or ex vivo expansion approaches centered on the homeostatic cytokine IL-15 were developed. Here, we describe the development of an ex vivo expansion system based on a feeder cell expressing membrane-bound IL-21 that enables log-phase growth of primary NK cells for many weeks without inducing senescence, and describe the biology, correlative science, and translation to clinical trials for patients with leukemia, brain tumors, and solid tumors.

Abstract LB-088: Cytokine-transgenic NOG mice engrafted with human peripheral blood cells support natural killer cell expansion

Abstract Harnessing natural killer (NK) cells and their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) forms the basis for many ongoing immuno-oncology efforts. Currently, most preclinical in vivo studies of ADCC-dependent efficacy rely on syngeneic models, which mandate the generation and use of surrogate therapies to overcome differences between mouse and human immune systems. These steps add time and cost, and can be particularly challenging for advanced antibody therapeutics (e.g. bispecific antibodies and antibody-drug conjugates). A humanized immune system (HIS) model that supports human NK cells could improve evaluations of human NK cell-dependent cancer therapies. However, HIS models have largely failed to support human NK cell-engraftment, rendering them inadequate for ADCC studies. Prior work has shown that human cytokine-transgenic hIL2-NOG and hIL15-NOG mice support the development of human NK cells from engrafted human hematopoietic stem cells (HSCs). Furthermore, the hIL15-NOG model has been shown to support in vivo expansion and long-term engraftment of isolated human NK cells. These properties suggest hIL2-NOG and/or hIL15-NOG might also sufficiently support NK cells following engraftment of peripheral blood mononuclear cells (PBMCs). Such a model could enable cost-effective and efficient in vivo studies of human NK cells and therapeutic human antibodies. To test this hypothesis, NOG, hIL-2 NOG, and hIL-15 NOG mice were engrafted with PBMCs from a single donor and at one of three increasing cell doses. The onset of graft vs host disease and peripheral blood human immune cell subsets were monitored through the duration of this study. Results showed that hIL15-NOG mice had a slightly diminished survival compared to conventional NOG. Nevertheless, hIL15-NOG survived up to 7 weeks after PBMC engraftment without any signs of graft vs host disease. Although hIL2-NOG mice showed the best engraftment rate for NK cells and other immune cell subpopulations, the overall survival was severely decreased post engraftment. Comparing NK cell-engraftment in hIL15-NOG and conventional NOG mice, we observed a tenfold increase of NK cell numbers in hIL15-NOG mice, independent of engrafted PBMC numbers, making it a highly suitable HIS model for studying NK cells. Citation Format: Paul Volden. Cytokine-transgenic NOG mice engrafted with human peripheral blood cells support natural killer cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-088.

Ambient mass spectrometry-based detection system for tumor cells in human blood

Circulating tumor cells (CTCs) are a rare cell population in peripheral blood that lead to distant metastasis in many types of cancer. Most current CTC-detection methods, including antibody-based detection, have shortcomings such as false-positive and false-negative findings. These limitations must be addressed to achieve CTC detection with high accuracy and reproducibility; and therefore, clinical utility. Herein, we propose a novel method of tumor cell detection in human blood samples using ambient mass spectrometry (MS). Probe electrospray ionization (PESI)-MS was used to obtain mass spectra containing 1,136 peaks from peripheral blood mononuclear cells (PBMCs) spiked with different numbers of SAS tumor cells by. Similarities between the spectral patterns of PBMCs spiked with different numbers of tumor cells, and those from PBMCs alone, were statistically reduced with increasing numbers of SAS tumor cells. Intriguingly, the influences of a few hundred tumor cells could be observed in the changes to the spectral peaks of PBMCs. Moreover, 16 individual peaks, with positive and negative correlations between peak intensities and the number of SAS tumor cells, were identified as tumor-associated metabolites. In conclusion, we successfully used PESI-MS to detect tumor cells present among very large numbers of PBMCs without the need for enrichment using antibodies for tumor cell markers. Our data strongly suggest that this method may be clinically suitable for CTC detection.

Semaphorin 7A in circulating regulatory T cells is increased in autosomal-dominant polycystic kidney disease and decreases with tolvaptan treatment.

Semaphorin 7A (SEMA7A) is an immunomodulating protein implicated in lung and liver fibrosis. In autosomal-dominant polycystic kidney disease (ADPKD), the progressive expansion of renal cysts, inflammation, and subsequent renal fibrosis leads to end-stage renal disease (ESRD). SEMA7A may play a role in renal fibrosis and in ADPKD. We evaluated Sema7a in a mouse model of renal fibrosis and determined the expression of SEMA7A in human ADPKD kidney. We analyzed SEMA7A expression on peripheral blood mononuclear cells (PBMCs), including CD45+ (leukocyte), CD14+(monocyte), CD4+ (T lymphocytes) and CD4+Foxp3+CD25+ [regulatory T lymphocytes (Tregs)] from 90 ADPKD patients (11 tolvaptan treated and 79 tolvaptan naïve), and 21 healthy volunteers, using a Fluorescence-Activated Cell Sorting (FACS). Sema7a is required for renal fibrosis. SEMA7A shows robust expression in ADPKD kidneys, localizing to cysts derived from distal tubules. SEMA7A is higher in circulating monocytes, but unchanged in CD4+ lymphocytes in ADPKD patients. The SEMA7A increase was detected early (stage 1 CKD) and seemed more prominent in patients with smaller kidneys (p = 0.09). Compared to tolvaptan-naïve ADPKD patients, those treated with tolvaptan showed reduced SEMA7A expression on monocytes, T lymphocytes, and Tregs, although the number of PBMCs was unchanged. After 1month of tolvaptan treatment, SEMA7A expression on Tregs decreased. SEMA7A shows potential as both a therapeutic target in mammalian kidney fibrosis and as a marker of inflammation in ADPKD patients. SEMA7A expression was lower after tolvaptan treatment, which may reflect drug efficacy.

A Modified Ficoll-Paque Gradient Method for Isolating Mononuclear Cells from the Peripheral and Umbilical Cord Blood of Humans for Biobanks and Clinical Laboratories.

Although the Ficoll-Paque method is classically used to isolate peripheral blood mononuclear cells (PBMCs), modifications in this method are required for a more rapid and economic output for biobanks and clinical laboratories, particularly in developing countries. In this study, we addressed this issue by modifying the Ficoll-Paque method for the isolation of PBMCs or mononuclear cells from the peripheral and the umbilical cord blood of healthy and diseased (infected, anemic, and chronic obstructive pulmonary disease) adult individuals. In the modified method, we initiated the cell isolation process from the buffy coat layer, which appears in the interface between the plasma and sediments after centrifugation, instead of using the whole blood as described in the classic method. Although the PBMC yield by the modified method was about 12% less than in the classic method, the number of PBMCs isolated by the modified method was more than one million, which is enough for different research/diagnostic purposes, such as multi-omics detection. Assessment of cell viability and purity by hematology analyzer and trypan blue showed no significant difference between the viability and purity of the PBMCs isolated by these two methods in almost all groups, except samples from the infected and cord blood groups, where lower PBMC purity with higher granulocyte contamination were observed. In addition, at delayed processing time points, all parameters for the two methods were decreased in a time-dependent manner, especially at 8, 12, or 24 hours after the sample collection. In summary, the performance of PBMC isolation by the classic and modified methods mainly relies on the PBMC ratio in original samples. The modified method could be preferred for PBMC isolation because of its time and cost savings, especially for the biobanks and clinical laboratories in developing countries.

Effect of mild heat stress on heat shock protein 70 in a balneotherapy model

IntroductionFever-induced mild heat stress plays an important role in cellular responses. The best-known heat shock response is heat shock protein (HSP) secretion. We investigated the effect of mild heat stress on serum HSP70 levels and a possible association between the immune system and these molecules by applying a mild heat stress model using a thermal water bath. MethodPeripheral blood samples from 16 osteoarthritis (OA) patients who were treated with 39–40°C thermo-mineral water immersion were examined. Samples were drawn before the first bath, after the first bath, and at the end of treatment. HSP70 levels and interferon-gamma (IFN)-γ responses in peripheral blood mononuclear cells (PBMC) were evaluated in plasma by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assay, respectively. ResultsIFN-γ-secreting PBMC numbers were increased significantly in response to HSP70, purified protein derivative (PPD), and phytohemagglutinin (PHA) stimulation after the first bath, as compared to before bathing values (p=0.005, p=0.012, p=0.021). HSP level in serum was also increased after the first bath, and decreased to near-baseline levels after treatment, however the differences were not significant. ConclusionsThe initial increase in HSP 70 and IFN-γ levels after the first bath and the final decrease at the end of serial baths suggest that the immune system adapts to the heat stress at the end of this intervention.