Use Of Peripheral Blood Mononuclear Cells Research Articles

PBMC-mediated modulation of macrophage polarization in RAW264.7 cells through STAT1/STAT6 signaling cascades

Peripheral blood mononuclear cells (PBMC), sourced autologously, offer numerous advantages when procured: easier acquisition process, no in vitro amplification needed, decreased intervention and overall increased acceptability make PBMC an attractive candidate for cell therapy treatment. However, the exact mechanism by which PBMC treat diseases remains poorly understood. Immune imbalance is the pathological basis of many diseases, with macrophages playing a crucial role in this process. However, research on the role and mechanisms of PBMC in regulating macrophages remains scarce. This study employed an in vitro co-culture model of PBMC and RAW264.7 macrophages to explore the role and mechanisms of PBMC in regulating macrophages. The results showed that the co-culturing led to decreased expression of inflammatory cytokines and increased expression of anti-inflammatory cytokines in RAW264.7 or in the culture supernatant. Additionally, the pro-inflammatory, tissue matrix-degrading M1 macrophages decreased, while the anti-inflammatory, matrix-synthesizing, regenerative M2 macrophages increased in both RAW264.7 and monocytes within PBMC. Moreover, co-cultured macrophages exhibited a significantly decreased p-STAT1/STAT1 ratio, while the p-STAT6/STAT6 ratio significantly increased. This suggests that PBMC may inhibit M1 macrophage polarization by blocking STAT1 signaling cascades and may promote M2 macrophage polarization through the activation of STAT6 signaling cascades. Overall, this study sheds light on the role and mechanism of PBMC in regulating macrophages. Moreover, it was found that monocytes within co-cultured PBMC differentiated into M2 macrophages in the presence of macrophages. This finding provides experimental evidence for the use of PBMC in treating inflammatory diseases, especially macrophage-depleting inflammatory diseases such as osteoarthritis.

236 Systemic mitochondrial dysfunction in monogenic Parkinson’s disease as a potential biomarker for stratification

To investigate the use of PBMCs as a biomarker of mitochondrial dysfunction in Parkinson disease (PD) fresh peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll gradient separation from PD patients with pathogenic biallelic parkin mutations as well as healthy controls (HC). PBMC mitochondrial function was analysed using Seahorse XF respirometry and flow cytometric assessment of mitochondrial membrane potential (Δψm) using TMRE staining.Nine parkin PD patients (average age 45.2yrs, 66% male) and 41 healthy controls (average age 57.8yrs, 58% female) were sampled for the study. Parkin patients had increased oxygen consumption related to proton leak, reduced maximal respiration and reduced spare respiratory after correction for age (p<0.01). Additionally Parkin patients were found to have reduced resting Δψm and reduced oxygen consumption related to ATP production (p<0.01). Basal oxygen consumption was similar between Parkin patients and healthy controls.These findings are in keeping with parkin’s role in removing dysfunctional mitochondria through mitophagy. The finding of normal basal oxygen consumption suggests that dysfunction is most pronounced when cells are under stress, during high ATP demand. This ‘signature’ of mitochondrial dysfunction could be applied to idiopathic PD patients to define a mitochondrial subgroup, in which personalised, stratified therapeutics could be targeted.p.campbell@ucl.ac.uk

Biomarkers in Human Peripheral Blood Mononuclear Cells: The State of the Art in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by the progressive loss of lower motor neurons, weakness and muscle atrophy. ALS lacks an effective cure and diagnosis is often made by exclusion. Thus, it is imperative to search for biomarkers. Biomarkers can help in understanding ALS pathomechanisms, identification of targets for treatment and development of effective therapies. Peripheral blood mononuclear cells (PBMCs) represent a valid source for biomarkers compared to cerebrospinal fluid, as they are simple to collect, and to plasma, because of the possibility of detecting lower expressed proteins. They are a reliable model for patients’ stratification. This review provides an overview on PBMCs as a potential source of biomarkers in ALS. We focused on altered RNA metabolism (coding/non-coding RNA), including RNA processing, mRNA stabilization, transport and translation regulation. We addressed protein abnormalities (aggregation, misfolding and modifications); specifically, we highlighted that SOD1 appears to be the most characterizing protein in ALS. Finally, we emphasized the correlation between biological parameters and disease phenotypes, as regards prognosis, severity and clinical features. In conclusion, even though further studies are needed to standardize the use of PBMCs as a tool for biomarker investigation, they represent a promising approach in ALS research.

Vitamin D status influences transcriptional levels of RANKL and inflammatory biomarkers which are associated with activation of PBMC

Vitamin D status is involved in the risk of many chronic diseases including cancer, inflammatory and autoimmune disease. The RANK/RANKL/OPG system is also implicated in the orchestration of immune functions.We aimed to investigate the expression of RANKL, OPG and markers of inflammation and immune activation in peripheral blood mononuclear cells (PBMC) from healthy subjects with different 25(OH)D3 plasma levels.The 25(OH)D3 plasma concentrations were assessed by HPLC. The gene expression was evaluated by qRT-PCR.The expression of CYP27B1 was lower in subjects with 25(OH)D3 levels below 50 nmol/L (deficiency) than subjects with both insufficient and sufficient levels of 25(OH)D3. In subjects with deficiency, we observed the up-regulation of RANKL, TNF-α, IFN-γ, ICAM and LFA-1, and a reduction of the anti-inflammatory cytokines IL-13 and IL-4 in comparison to other subjects. Finally, we found a negative correlation between RANKL mRNA levels and 25(OH)D3 and between 25(OH)D3 and ICAM mRNA levels. A positive correlation between ICAM and RANKL was observed.Our results give evidence of the modulatory effects of circulating 25(OH)D3 levels on gene expression of biomarkers of immune activation in PBMC, suggesting the possible use of PBMC as ex-vivo model to characterize molecular mechanisms of immune/inflammatory response in hypovitaminosis conditions.

Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells

BackgroundClinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes.MethodsCounter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products.ResultsWhen compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells.ConclusionsEnrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

Porcine antigen-specific IFN-γ ELISpot as a potentially valuable tool for monitoring cellular immune responses in pig-to-non-human primate islet xenotransplantation.

Recent progress in xenotransplantation of porcine islets to non-human primates (NHPs) gives hope for human clinical trials in the near future. Thus, implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen-specific cellular immune responses is necessary. The enzyme-linked immunospot (ELISpot) assay has been widely used to monitor antigen-specific alloreactive T-cell responses in humans; however, the utility of porcine islet-specific ELISpot assay has not yet been thoroughly evaluated for pig-to-NHPs intraportal islet xenotransplantation. The optimal ELISpot assay conditions, including the number of responder and stimulator cells and the provision of costimulation, were determined. Then, ELISpot assays were conducted on serial stocks of peripheral blood mononuclear cell (PBMC) samples previously isolated from NHP recipients transplanted with porcine islets. Either splenocytes from donor pigs or pancreatic islets from third-party pigs were used for antigen stimulation. At the same time, the ratio of CD4(+) /CD8(+) T cells and the percentage of CD4(+) FoxP3(+) T cells in the peripheral blood were evaluated. Finally, liver biopsy samples were evaluated to assess the immunopathology of the grafts. The optimal conditions for the ELISpot assay were defined as 2.5 × 10(5) responder cells incubated with 5.0 × 10(5) stimulator cells in 96-well, flat-bottom plates without further costimulation. Using donor splenocytes as stimulators, a serial interferon-gamma (IFN-γ) ELISpot assay with PBMCs from the monkeys with prolonged porcine islet grafts (>180 days) demonstrated that the number of donor antigen-specific IFN-γ-producing cells significantly increased upon overt graft rejection. However, use of third-party porcine islets as stimulators did not reflect graft rejection, suggesting that the use of donor-specific PBMCs, and not tissue (porcine islet)-specific cells, as stimulators could better serve the purpose of this assay in adult porcine islet transplantation. IFN-γ spot number was neither influenced by the peripheral blood CD4(+) /CD8(+) T-cell ratio nor the percentage of CD4(+) FoxP3(+) T cells. Finally, in cases of overt graft rejection, the number of IFN-γ spots and the graft-infiltrating T cells in biopsied liver samples increased simultaneously. Use of PBMCs in a porcine antigen-specific IFN-γ ELISpot assay is a reliable method for monitoring T-cell-mediated rejection in pig-to-NHP islet xenotransplantation.

Socializing Individualized T-Cell Cancer Immunotherapy.

Tumors typically accumulate hundreds of mutations in “driver” genes critical to transformation as well as in other “passenger” genes.1 These mutations can generate neoantigens—mutated versions of normal proteins that become recognizable by patients’ own T cells.2,3 Such T cells recovered from patients have been used as a source of tumor-hunting T-cell therapies, representing the epitome of individualized medicine as a way to treat tumors.4,5 Unfortunately, at best only a very small handful of potential neoantigens are recognized by a patient’s own T cells,6 probably because of the unrelenting immunosuppressive tactics exerted chronically on the patient’s own T-cell repertoire. Now, Stronen et al. have shown that T cells from normal, healthy donors can recognize many more neoantigens from a given patient than the patient’s own T-cell repertoire allows.7 A situation in which a greater number of neoantigens are recognized by more T cells can only be good news for the treatment of malignant tumors. These results suggest that even individualized cancer immunotherapy might benefit from reaching out into the community for extra T-cell help.

187 - Use of Elutriated Lymphocytes for Manufacturing CD19-Chimeric Antigen Receptor T Cells Improves the Quantity of Transduced T Cells in the Final Product

Clinical trials of CD19-Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of B cell lymphoma and leukemia have been promising. Yields of CD19-CAR T cells have been variable and we have shown that this variability is due in part to the contamination of the PBMCs used as starting cellular material with monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using counter-flow elutriation for CD19-CAR T cell manufacturing. Elutriation allows for the rapid, closed system separation of lymphocytes from monocytes and granulocytes. CD19-CAR T cells were manufactured from 26 patients using autologous PBMCs, anti-CD3/CD28 magnetic beads to promote T cell expansion, two days of retroviral transduction and 7 days of culture. For 6 products lymphocyte enrichment was performed using anti-CD3/CD28 beads, for 15 plastic adherence and anti-CD3/CD28 bead selection and for 5 elutriation. One product manufactured with anti-CD3/CD28 bead selected T cells did not contain enough cells for one clinical dose (1 × 106 transduced cells per kg). Among the other 25 products all were rich in T cells (>93%) and had high transduction efficiency (32% to 90%). The quantity of transduced T cells in the final product was greatest for those manufactured with elutriated lymphocytes (581 ± 212 × 106 cells/kg) and was the least for products manufactured with anti-CD3/CD28 bead selected cells (253 ± 227 × 106 cells/kg; p < 0.04). Products manufactured from plastic adherence and anti-CD3/CD28 selected cells had an intermediated quantity of transduced T cells (393 ± 176 × 106 cells/kg). In conclusion, enrichment of PBMCs for lymphocytes using elutriation increases the quantity of transduced cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

Taking advantage of an old concept, "illegitimate transcription", for a proposed novel method of genetic diagnosis of McArdle disease.

McArdle disease is a metabolic disorder caused by pathogenic mutations in the PYGM gene. Timely diagnosis can sometimes be difficult with direct genomic analysis, which requires additional studies of cDNA from muscle transcripts. Although the "nonsense-mediated mRNA decay" (NMD) eliminates tissue-specific aberrant transcripts, there is some residual transcription of tissue-specific genes in virtually all cells, such as peripheral blood mononuclear cells (PBMCs). We studied a subset of the main types of PYGM mutations (deletions, missense, nonsense, silent, or splicing mutations) in cDNA from easily accessible cells (PBMCs) in 12 McArdle patients. Analysis of cDNA from PBMCs allowed detection of all mutations. Importantly, the effects of mutations with unknown pathogenicity (silent and splicing mutations) were characterized in PBMCs. Because the NMD mechanism does not seem to operate in nonspecific cells, PBMCs were more suitable than muscle biopsies for detecting the pathogenicity of some PYGM mutations, notably the silent mutation c.645G>A (p.K215=), whose effect in the splicing of intron 6 was unnoticed in previous muscle transcriptomic studies. We propose considering the use of PBMCs for detecting mutations that are thought to cause McArdle disease, particularly for studying their actual pathogenicity.Genet Med 18 11, 1128-1135.

Clinical laboratory experience of blood CRIM testing in infantile Pompe disease

Cross-reactive immunological material (CRIM) status is an important prognostic factor in patients with infantile Pompe disease (IPD) being treated with enzyme replacement therapy. Western blot analysis of cultured skin fibroblast lysates has been the gold standard for determining CRIM status. Here, we evaluated CRIM status using peripheral blood mononuclear cell (PBMC) protein. For 6 of 33 patients (18%) CRIM status determination using PBMC was either indeterminate or discordant with GAA genotype or fibroblast CRIM analysis results. While the use of PBMCs for CRIM determination has the advantage of a faster turnaround time, further evaluation is needed to ensure the accuracy of CRIM results.

Peripheral blood mononuclear cells as a potential source of biomarkers to test the efficacy of weight-loss strategies.

Peripheral blood mononuclear cells (PBMC) constitute an easily obtainable blood cell fraction useful in nutrition and obesity studies. Our aim was to study the potential use of PBMC to reflect metabolic recovery associated with weight loss in rats. By real-time PCR, the fasting response of key energy homeostatic genes in PBMC samples of control and cafeteria-obese rats and of rats fed a control diet after the intake of a cafeteria diet (post-cafeteria model) was analyzed. Fasting caused decreased mRNA expression of lipogenic (Fasn and Srebp1a) and adipogenic (Pparγ) genes in PBMC, whereas it increased the expression of the key beta-oxidation gene Cpt1a and the orexigenic gene Npy. Fasting response of the genes studied was impaired in cafeteria-obese animals but was recovered in post-cafeteria rats, which showed a significant body weight decrease and normalization of adipose and metabolic parameters. Npy expression analyzed in PBMC has been revealed to be especially useful as a marker of fasting sensitivity, as its fasting response is not affected by the age of the animals and it is recovered even after shorter time of exposure to a balanced diet. PBMC reflect homeostatic balance recovery associated with weight loss in obese animals, when reverting from a hyperlipidic to a control balanced diet.

Whole-exome sequencing of DNA from peripheral blood mononuclear cells (PBMC) and EBV-transformed lymphocytes from the same donor

BackgroundThe creation of lymphoblastoid cell lines (LCLs) through Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings.ResultsWe performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs.ConclusionsBy varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.

Validation of the Use of Peripheral Blood Mononuclear Cells as Surrogate Model for Skeletal Muscle Tissue in Nutrigenomic Studies

Peripheral blood mononuclear cells (PBMCs) offer a significant promise for gene expression analyses as a substitute for tissues that are not easily accessible. The objective of this study was to validate the use of PBMCs for gene expression analysis as a marker of nutritional intervention as an alternative to skeletal muscle tissue (SMT) biopsies. We performed a transcriptome comparison of PBMCs versus SMT after an 8-week supplementation with n-3 polyunsaturated fatty acid (PUFA) in 16 obese and insulin-resistant subjects. Expression levels of 48,803 transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina, San Diego, CA). In SMT, 36,738 (75%) transcripts were detected, whereas 34,182 (70%) transcripts were detected in PBMCs. Further, 88% (32,341) of these transcripts were coexpressed in both tissues. Importantly, a strong correlation (r = 0.84, p < 0.0001) was observed between transcript expression levels of PBMCs and SMT after n-3 PUFA supplementation. In conclusion, PBMCs express the majority of transcripts expressed in SMT subsequent to n-3 PUFA supplementation and their expression levels are comparable. In the interest of practicalities and cost, these results support the use of PBMCs as a surrogate model for SMT gene expression in nutrigenomic studies. Further research on PBMC and SMT gene expression in response to other nutritional exposures is warranted.

Effect of PBMC shipment and storage conditions on ELISpot activity

INTRODUCTION: Cellular immune response is often evaluated on frozen Peripheral Blood Mononuclear Cells (PBMC). Here, we evaluate the effect of shipping and storage conditions on PBMC viability and functional response using ELISpot assay.METHODS: Cryopreserved human PBMC were evaluated after overnight placement on dry ice and subsequent storage at −80oC, −20°C, in LN2 and on dry ice for up to 6 months. Cell count and viability was measured at time of thaw and after 24 hour rest. ELISpot assays were performed using huIFN‐γ ELISpot Kit (SeraCare) after stimulation with PHA, CMV and CEF peptide pools.RESULTS: Cells stored overnight on dry ice showed no significant decrease in viability at thaw and post rest. Cells stored at −20°C were not suitable for ELISpot assay. Cells stored on dry ice, at −80°C or in LN2 for 6 month demonstrated 92–93% viability upon thaw and 80–86% after 24 hr rest. The control cells remained 90% viable both post thaw and post rest. ELISpot responses of cells stored at −80°C or in LN2 for 6 month, or cells stored on dry ice for 4 weeks were comparable to control.CONCLUSIONS: The PBMC shipped in dry ice can be stored at −80°C for up to 6 months or on dry ice for 4 weeks with no appreciable loss of viability or functionality. Therefore, the use of PBMC for ELISpot proficiency testing is possible even in laboratories which lack ultra‐cold storage facilities.SUPPORT: This work was supported by NIAID contract NO1‐AI‐85341.

Modeling partially effective HIV vaccines in vitro.

Significant public health benefits could be realized with human immunodeficiency virus (HIV) vaccines that are incompletely effective. However, standard assays of experimental HIV vaccine immunogenicity may not correlate with antiviral effectiveness and cannot identify subtle effects. We developed an in vitro challenge assay (IVCA) that measures the net antiviral effect in whole peripheral blood mononuclear cells (PBMCs) to any titered HIV isolate. We then modeled partially effective postvaccination immune status 4 ways: use of PBMCs from highly exposed, uninfected individuals; depletion and partial reconstitution of autologous CD8+ cells from PBMCs from HIV-positive long-term nonprogressors; partial blocking of infection with chemokines; and variation in challenge virus dose. IVCA could detect as little as 3-fold differences in the challenge titer (30, 10, and 3 50% tissue-culture infective doses) or odds ratio of HIV infection. This robust and simple assay should be useful in determining which HIV vaccine candidates are suitable for field trials of efficacy.

Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.

The Distribution and Duration of Hantaan Virus in the Body Fluids of Patients with Hemorrhagic Fever with Renal Syndrome

The distribution and duration of Hantaan virus (HTNV) in the body fluids of patients were studied by immunofluorescence, reverse passive hemagglutination, and cell culture assays. Virus antigen of hemorrhagic fever with renal syndrome in peripheral blood mononuclear cells (PBMCs) was usually present before day 11 of the disease, especially from days 4-7. Virus isolates were more readily recovered from plasma early in the course of the illness and less frequently after day 7. The use of PBMCs rather than plasma enabled isolates to be recovered at a rate nearly twice that permitted by plasma and allowed the isolation peak of HTNV (days 4-7 after onset of disease) to extend an additional 2 or 3 d, thus prolonging the period of detectable viremia until days 8-11. PBMCs were especially useful in isolating viruses from patients with hemorrhagic fever with renal syndrome in whom antibody titers were generally high during the acute phase of the disease. HTNV was isolated from the cerebrospinal fluid of patients, but was difficult to recover from other body fluids.