Frozen Peripheral Blood Mononuclear Cells Research Articles

Anti-inflammatory genes in PBMCs post-spontaneous intracerebral hemorrhage.

BackgroundNeuroinflammation is important in the pathophysiology of spontaneous intracerebral hemorrhage (ICH) and peripheral inflammatory cells play a role in the clinical evolution and outcome.MethodologyBlood samples from ICH patients (n = 20) were collected at admission for 5 consecutive days for peripheral blood mononuclear cells (PBMCs). Frozen PBMCs were used for real-time PCR using Taqman probes (NFKB1, SOD1, PPARG, IL10, NFE2L2, and REL) and normalized to GAPDH. Data on hospital length of stay and modified Rankin score (MRS) were collected with 90-day MRS ≤ 3 as favorable outcome. Statistical analysis of clinical characteristics to temporal gene expression from early to delayed timepoints was compared for MRS groups (favorable vs unfavorable) and hematoma volume.Principle findings and resultsIL10, SOD1, and REL expression were significantly higher at delayed timepoints in PBMCs of ICH patients with favorable outcome. PPARG and REL increased between timepoints in patients with favorable outcome. NFKB1 expression was not sustained, but significantly decreased from higher levels at early onset in patients with unfavorable outcome. IL10 expression showed a negative correlation in patients with high hematoma volume (>30 mL).Conclusions and significanceAnti-inflammatory, pro-survival regulators were highly expressed at delayed time points in ICH patients with a favorable outcome, and IL10 expression showed a negative correlation to high hematoma volume.

Establishment and Preservation of Lymphoblastoid Cell Lines from Fresh and Frozen Whole Blood and Mononuclear Cells.

Although blood cells are interesting sources for genome investigations, one of the main problems in obtaining genomic DNA from blood is the restricted amount of DNA. This obstacle can be avoided by generating Epstein-Barr virus (EBV)-induced B cell lines. This study investigates the efficiency of four different methods to generate lymphoblastoid cell lines (LCLs). Blood samples (n = 120) were obtained from donors and categorized into four groups: fresh whole blood, frozen whole blood, fresh peripheral blood mononuclear cells (PBMCs), and frozen PBMCs. The samples were followed by EBV transformation to generate LCLs. Quality control and authentication of the cells were performed using multiplex PCR and short tandem repeat (STR) analyses. Finally, we assessed the success rate and amount of time to establish the cell lines in each group. The results showed that the cells were not contaminated nor were they misidentified or cross-contaminated with other cells. The success rate of LCLs generated from the whole blood groups was lower than the PBMC groups. The freezing procedures did not have any considerable effect on the establishment of lymphoblastoid cells. These established cells have been preserved in the human and animal cell bank of the Iranian Biological Resource Center (IBRC) and are available for researchers. Due to the management and transformation of a substantial number of blood samples, we recommend that researchers freeze PBMCs for further use with high efficiency and time-saving. We suggest that whole fresh blood should be directly transformed when the volume of the blood sample is less than 0.5ml.

Early reduction of regulatory T cells is associated with acute rejection in liver transplantation under tacrolimus-based immunosuppression with basiliximab induction.

Regulatory T (Treg) cells are important in preventing acute rejection (AR) in solid organ transplantation, but the clinical relevance of the different kinetics early after liver transplantation (LT) in acute rejectors and non-rejectors is unclear. We analyzed peripheral blood samples of 128 LT recipients receiving basiliximab induction plus tacrolimus immunosuppression. Samples were obtained at pretransplant, D7, and D30 after LT. Frequency and phenotype of Tregs were analyzed by flow cytometry. The predictive value of Treg frequency at D7 was assessed for suspected acute rejection (SAR) and was validated for biopsy-proven AR (BPAR). We found that the frequencies of total and activated Tregs at D7 were significantly lower in recipients with SAR and BPAR. Treg was more reduced in BPARs by in vitro tacrolimus treatment in the presence of basiliximab. Moreover, an early reduction of Treg frequency in rejectors was associated with a greater increase in Treg apoptosis and further attenuated IL-2 signaling. D7 Treg frequency was an independent risk factor for SAR, which was also validated for BPAR. In conclusion, first-week peripheral blood Treg frequency correlates with AR after LT under tacrolimus-based immunosuppression, which needs to be proven in larger, geographically and clinically diverse populations.

1381. M.genavense in the ART Era: From Persistent Disseminated Disease to Severe Disease

Background Mycobacterium genavense is an opportunistic pathogen in HIV patients that is difficult to culture and difficult to manage clinically. Here we describe three cases of HIV patients with Mycobacterium genavense with courses representing the spectrum of M.genavense presentations in the current ART era complicated by differing divergent immune responses.MethodsTwo patients were in a longitudinal study at NIAID enrolling patients with HIV and suspected IRIS (PANDORA) (NCT02147405) and one was seen at St. James’s hospital in Dublin. Frozen peripheral blood mononuclear cells were collected at the time of presentation and were used for in vitro stimulation with irradiated M. genavenese in cases 1 and 2 to detect production of cytokines by CD4 T cells.ResultsPt 1: 27-year old male with M. genavense presenting as diarrhea, abdominal pain, skin nodules, hepatosplenomegaly, and lymphadenopathy (LAN) that persisted on one year of anti-mycobacterial therapy and ART. No CD4 T-cell cytokine response to M. genevense genavense was detected (Fig 1). He received interferon-g and optimization of his antimycobacterial regimen with improvement of symptoms and decreased pathogen burden on repeated biopsies. Pt 2: 55-year old female with M. genavense IRIS manifesting as fevers and abdominal pain that persisted for 10 months on ART (CD4 109 cells/µmL) requiring intermittent corticosteroid use complicated by adrenal insufficiency. She had evidence of CD4 T-cell response to M. genavense in vitro and improved with optimization of her anti-mycobacterial and corticosteroid regimen. Pt 3: 39-year-old male with M. genavense IRIS presenting as fevers, LAN, pleuritic chest pain, and abdominal pain on ART (CD4 19 c/mµL) persisting despite immunologic response to ARV therapy (CD4 recovery to 419 c/mL), appropriate anti-mycobacterial therapy and corticosteroids. He required 4 doses of infliximab (5 mg/kg IV) that facilitated tapering of prednisone.ConclusionThe clinical presentation of Mycobacterium genavense in HIV patients in the ARV era range from disseminated disease with poor immune reconstitution to persistent or severe IRIS requiring immune suppression. Effective clinical outcomes relied on appropriate anti-mycobacterial and either immune-boosting or immune-suppressive therapies. Disclosures All authors: No reported disclosures.

Increased KLRG1 and PD-1 expression on CD8 T lymphocytes in TB-IRIS.

BackgroundTuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory complication in HIV-TB co-infected patients receiving antiretroviral therapy (ART). The exact contribution of T cells, natural killer (NK) cells, and monocytes to TB-IRIS development remains unclear. Here, we studied the expression of exhaustion markers on lymphocytes at different intervals during ART.MethodsWe compared 13 HIV-TB patients who developed TB-IRIS with 13 patients who did not (HIV+TB+), 13 HIV-patients without TB (HIV+TB-) and 9 HIV/TB-negative controls (HIV-TB-). Patients did not differ in age, gender, or CD4-count prior to ART. Frozen peripheral blood mononuclear cells, collected before ART and during 3 months and 9 months of ART, were analysed using flow cytometry. We examined expression of KLRG1, PD-1 and IL-27R on CD4+ and CD8hi T cells, as well as CD3-negative CD8lo lymphocytes as an approximate subset of NK cells. In addition, expression of TLR2, TLR4, IL1RL1, and TRAILR on CD14+ monocytes were investigated.ResultsPrior to ART, TB-IRIS patients had higher percentages of CD8hi T cells that are KLRG1+PD-1+ compared to each control group (p≤0.034). Though PD-1 expression decreased during ART in all groups (p≤0.026), the percentage KLRG1+PD-1+CD8hi T cells remained higher in TB-IRIS patients after 3 months of ART (p≤0.013). Though these patterns were less pronounced in CD3-CD8lo lymphocytes, the percentage of KLRG1+ cells was higher in TB-IRIS patients prior to ART (p≤0.043). In contrast, no clear differences could be observed for CD4+ T cells or monocytes.ConclusionTB-IRIS is preceded by a high level of exhausted (KLRG1+PD-1+) CD8hi T cells, which persists during 3 months of ART. This trait is potentially mirrored in a subpopulation of NK cells, but not CD4+ T cells. Since a dysfunctional CD8+ lymphocyte compartment could predispose patients to TB-IRIS, the functional role of these cells prior to TB-IRIS development should be further explored.

A Simplified Method for Transduction and Expansion of T Cells for Clinical Application

Introduction: Genetically modified T cells are being investigated to treat a variety of disorders and have been particularly successful in treating B cell cancers. As more effort is poured into new targets, molecular switches, and various other modifications, development of processes to quickly manufacture new products must keep up. Current manufacturing processes often require highly skilled operators and specialized equipment. Here, we demonstrate a simplified, novel method for transduction of T cells, followed by robust expansion in the G-Rex bioreactor with no need for intervention until harvest. A scaled-up, closed-version of the same process, including a closed harvest step with the GatheRex is currently under evaluation.Methods: Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors were used as starting material. PBMCs were thawed, washed, and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags (32-C, Saint-Gobain Cell Therapy) or in G-Rex bioreactors (Wilson Wolf Corporation). Cells were cultured in TexMACS GMP medium (Miltenyi Biotec) with IL-7 and IL-15 throughout. For the transduction step, activated PMBCs and retroviral supernatant were incubated in cell culture bags coated with Retronectin (Takara) or in G-Rex bioreactors with vectofusin-1 (Miltenyi Biotec). Viral constructs contained either a CD34 or CD19 marker detectable by antibody staining. For transduction in the G-Rex, various cell densities, volumes, constructs, and multiplicity of infection (MOIs) were tested. Where applicable, the GatheRex device (Wilson Wolf Corporation) was used for volume reduction and harvest. Transduction efficiency and T cell phenotype were measured by flow cytometry. Cell count and viability were assessed with the NC-3000 (Chemometic). Glucose and lactate concentrations were checked daily for in-processing monitoring.Results: Overall transduction efficiency ranged from 30-90% depending on the experimental conditions. Incubating 1x107 activated PBMCs in 10 ml of medium in a 10-cm2 G-Rex (1.0 ml/cm2) with retrovirus at an MOI of 1 resulted in 3% transduced cells. Addition of vectofusin-1 to the same condition yielded transduction efficiency of 44%. Increasing the MOI to 10 lead to 86% transduced cells. Decreasing the transduction volume from 1.0 ml/cm2 to 0.4 ml/cm2 increased transduction efficiency from 34% to 55%. Reducing the volume further to 0.2 ml/cm2 did not improve efficiency, and rather had a negative impact compared to the 0.4 ml/cm2 condition (38%). 16-24 hrs after transduction, the volume of medium was increased to 10.0 ml/cm2 without a wash step. The dilution in place of a wash step had no negative impact on cell viability. 10.0 ml/cm2 medium supported high viability (>90%) and expansion (30-50 fold) over an additional 9 days without operator intervention. The phenotype of cells expanded in the G-Rex contained a mixed population of CD45RO+ and CD45RA+ cells, with a similar distribution of naive and memory cell subsets in G-Rex and bag cultures. Harvest of cells with the GatheRex was efficient; a 1L volume was reduced 10-fold in 5 minutes, and 95% of cells were recovered.Summary: T cells can be transduced with retroviral vectors in the G-Rex bioreactor. Clinically relevant levels of transgene expression can be achieved by combining reagents in the G-Rex, without complicated coating steps or time-consuming spinning steps. This simplified procedure reduces the hands-on time of the T cell transduction to minutes rather than hours. Transgenic cells can be expanded 30-50-fold in the G-Rex with limited operator intervention and without specialized equipment. DisclosuresKhalil:Bellicum Pharmaceuticals: Employment. Foster:Bellicum: Employment, Equity Ownership. Gagliardi:Bellicum Pharmaceuticals: Employment.

A probiotic modulates the microbiome and immunity in multiple sclerosis.

Effect of a probiotic on the gut microbiome and peripheral immune function in healthy controls and relapsing-remitting multiple sclerosis (MS) patients. MS patients (N = 9) and controls (N = 13) were orally administered a probiotic containing Lactobacillus, Bifidobacterium, and Streptococcus twice-daily for two months. Blood and stool specimens were collected at baseline, after completion of the 2-month treatment, and 3 months after discontinuation of therapy. Frozen peripheral blood mononuclear cells (PBMCs) were used for immune cell profiling. Stool samples were used for 16S rRNA profiling and metabolomics. Probiotic administration increased the abundance of several taxa known to be depleted in MS such as Lactobacillus. We found that probiotic use decreased the abundance of taxa previously associated with dysbiosis in MS, including Akkermansia and Blautia. Predictive metagenomic analysis revealed a decrease in the abundance of several KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways associated with altered gut microbiota function in MS patients, such as methane metabolism, following probiotic supplementation. At the immune level, probiotic administration induced an anti-inflammatory peripheral immune response characterized by decreased frequency of inflammatory monocytes, decreased mean fluorescence intensity (MFI) of CD80 on classical monocytes, as well as decreased human leukocyte antigen (HLA) D related MFI on dendritic cells. Probiotic administration was also associated with decreased expression of MS risk allele HLA-DQA1 in controls. Probiotic-induced increase in abundance of Lactobacillus and Bifidobacterium was associated with decreased expression of MS risk allele HLA.DPB1 in controls. Our results suggest that probiotics could have a synergistic effect with current MS therapies. Ann Neurol 2018.

Abstract 632: Genome-scale neoantigen screening using ATLAS™ prioritizes candidate antigens for immunotherapy in a non-small cell lung cancer patient

Abstract Despite the unprecedented efficacy of checkpoint blockade (CPB) therapy in treating some cancers, the majority of patients fail to respond. Several lines of evidence support that the combination of CPB and neoantigen vaccine prolongs survival curves in cancer patients. Capitalizing on neoantigens derived from non-synonymous somatic mutations is a good strategy for therapeutic immunization. Current approaches to neoantigen prioritization involve mutanome sequencing, in silico epitope prediction algorithms, and experimental validation of cancer neoepitopes. Even the best in class in silico epitope prediction algorithms lack the accuracy necessary for efficacious personalized cancer vaccines. We sought to circumvent some of the limitations of currently available prediction algorithms by prioritizing neoantigens empirically using ATLAS™, a technology developed to screen T cell responses from any subject against their entire complement of potential neoantigens. Exome sequences were obtained from peripheral blood mononuclear cells (PBMC) and tumor biopsies from a non-small cell lung cancer patient who had been successfully treated with pembrolizumab. The tumor exome was sequenced and somatic mutations were identified. Individual DNA sequences (399 nucleotides) spanning each mutation site were built, cloned and expressed in E. coli co-expressing listeriolysin O. Polypeptide expression was validated using a surrogate T cell assay or by Western Blotting. Frozen PBMCs, collected pre- and post-therapy, were used to derive dendritic cells (MDDC). Both CD4+ and CD8+ T cells were enriched and expanded using microbeads. The E. coli clones were pulsed onto MDDC in an ordered array, then co-cultured either with CD8+ or with CD4+ T cells overnight. T cell activation was detected by analyzing cytokines in supernatants. Antigens were identified as clones that induced a cytokine response that exceeded three standard deviations of the mean of all negative control wells, then their identities compared with T cell epitopes predicted using previously described algorithms. We found biological evidence for neoantigens that were specifically responsive to peripheral CD8+ and CD4+ T cells, derived from the patient’s tumor, pre- and post-CPB intervention. Some of these neoantigens were identified as a T cell target both pre- and post-CPB therapy. We identified neoantigens for which no epitopes were predicted by in silico methods. These data represent evidence that multiple patient-specific neoantigens can be identified through functional evidence of T cell response from peripheral blood without epitope prediction. By profiling natural and CPB-enhanced immunity to neoantigens, a broad catalog of T cell targets can be identified for development of immunotherapies that engage T cells against cancer to improve outcomes for patients for whom current therapies are ineffective. Citation Format: Lila Ghamsari, Emilio Flano, Judy Jacques, Biao Liu, Zheng Yan, Aula Alami, Christine Kelley, Theresa Zhang, Jonathan Havel, Vladimir Makarov, Taha Merghoub, Jedd D. Wolchok, Matthew Hellman, Pamela Carroll, Timothy Chan, Jessica B. Flechtner. Genome-scale neoantigen screening using ATLAS™ prioritizes candidate antigens for immunotherapy in a non-small cell lung cancer patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 632. doi:10.1158/1538-7445.AM2017-632

Anti-Thymocyte Globulin Kills Leukemic Stem Cells in Vitro

Background: Although curative treatment for acute myeloid leukemia, the success of allogeneic hematopoietic cell transplantation (HCT) is limited due to leukemia relapse and graft-versus-host disease (GvHD). Rabbit anti-thymocyte globulin (ATG) used for GvHD prophylaxis does not increase relapse (Walker et al: Lancet Oncol 2016). The non-increase in relapse could be because ATG has a direct anti-leukemic effect (Dabas et al: BBMT 2016). Multiple studies have suggested that a high number of leukemic stem cells (LSCs, also called leukemia initiating cells) remaining after therapy is associated with relapse. Therefore, targeting LSCs may be important for treating or preventing relapse. We demonstrated ATG's cytotoxic effect against acute myeloid leukemia (AML) blasts (Dabas et al: BBMT 2016). However, ATG's effect on LSCs has not been evaluated. In this study, we investigated in vitro ATG-induced complement-independent cytotoxicity (CIC, presumably direct induction of apoptosis) and complement-dependent cytotoxicity (CDC) against LSCs. This was also compared to ATG-induced CIC and CDC against healthy hematopoietic stem cells (HSCs).Methods:Frozen peripheral blood mononuclear cells (PBMNCs) from 15 patients newly diagnosed with AML were used as the source of LSCs. Frozen mononuclear cell apheresis samples from 15 healthy stem cell donors were used as the source of HSCs. We measured by flow cytometry CIC and CDC induced by ATG at 10 mg/L (the median peak concentration achieved with 4.5 mg/kg ATG given over day -2, -1 and 0) and 50 mg/L. CIC was induced by incubating cells with ATG in the presence of heat-inactivated (complement-depleted) fetal bovine serum for 4 hours. As a negative control for the calculation of background, cells were cultured under the same conditions except without ATG. CDC was induced by incubating cells with ATG in the presence of human serum as a source of complement for 15 minutes. As a negative control (for background), cells were incubated with ATG in the presence of heat-inactivated human serum (no complement). The LSCs and HSCs were phenotypically defined as CD45dim/neg, side scatterlow, CD34+, CD38neg and negative for CD14, CD16, CD19, CD56, CD235a and CD41a. For CIC, dying/dead cells were identified as Annexin V positive (Annexin V+). For CDC, dead cells were identified as 7-amino-actinomycin D positive (7AAD+).Results: ATG induced both CIC and CDC of LSCs at the concentration of 50 mg/L but not 10 mg/L. For CIC, the median percent Annexin V+ LSCs after incubation with 50 mg/L ATG was 22.3% vs 18.4% for background (p=0.0043, Wilcoxon matched pairs test). For CDC, the median percent 7AAD+ LSCs after incubation with 50 mg/L ATG was 37.2% vs 2% background (p=0.0004, Wilcoxon matched pairs test). Next, we compared the cytotoxicity of 50 mg/L ATG against LSCs versus healthy HSCs. For CIC, a significantly greater percent of LSCs than HSCs was killed (P=0.0363, Mann-Whitney rank sum test) (Figure 1). Similarly, for CDC, there was a trend toward a greater percent of LSCs than HSCs killed (P=0.0971) (Figure 2).Conclusion: ATG kills LSCs in vitro via both CIC and CDC. However, the degree of the killing is minor and only observed at a higher ATG concentration than typically achieved in patients. Our data also suggest that if high dose ATG was used in patients (resulting in ≥50 mg/L concentration), LSCs could be killed to a greater degree than healthy HSCs. [Display omitted] [Display omitted] DisclosuresDaly:Sanofi-Genzyme: Other: Advisory Board.

Detection and Characterization of Circulating Tumour Cells from Frozen Peripheral Blood Mononuclear Cells

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Detection and Characterization of Circulating Tumour Cells from Frozen Peripheral Blood Mononuclear Cells.

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker characterization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed “fresh” via Epic CTC Platform or from “frozen” PBMC pellets. Samples were analysed for CTC enumeration and biomarker characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC morphology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospectively collected samples.

Drug‐associated DNA Damage in HIV‐1 Patients taking HAART

Despite the reduction in mortality in HIV‐1 positive patients due to the use of highly active antiretroviral therapy (HAART), adverse effects have been associated to long‐term usage. Recently, several studies have demonstrated that HIV‐1 infected patients are under chronic oxidative stress caused by an inflammatory response to the latent infection, or as an adverse effect of the antiretroviral therapy. The aim of this study was to evaluate the DNA damage in peripheral blood mononuclear cells of HIV‐1 patients treated with HAART when compared to healthy donors. Frozen peripheral blood mononuclear cells (PBMCs) obtained from HIV‐1 positive subjects with undetectable viremia and under HAART were used for this study. An aliquot of the thawed cells was used for DNA isolation and the remaining cells were cultured for seven days. Single and double‐strand DNA breaks were evaluated using an alkaline gel electrophoresis (single‐strand gel electrophoresis) and the neutral comet assay, respectively. PBMCs from healthy donors were used as negative controls. EMS (ethylmethane sulfonate) was used as a positive control. Interestingly, the alkaline gel showed higher DNA fragmentation in HIV‐1 positive samples when compared to an HIV‐negative control and to a sample treated with EMS. Additionally, the neutral comet assay showed presence of double‐stranded DNA breaks in HIV‐1 positive cells when compared to a healthy donor sample. The extent of the single or double‐stranded DNA breaks varied among samples depending on type of therapy and the length of time under the therapy. Taken together, our results suggest that HAART may affect the oxidative stress imbalance in HIV‐1 positive patients.

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells' responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells' responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

The LINA cohort: indoor chemical exposure, circulating eosinophil/basophil (Eo/B) progenitors and early life skin manifestations

Hematopoietic progenitor cells, especially those committed to the Eo/B lineage, are known to contribute to allergic inflammation. The aim of the present study was to investigate whether environmental factors are associated with changes in numbers of circulating Eo/B progenitors at 1year of age. Peripheral blood from 60 1-year-old children enrolled in the LINA (Lifestyle and environmental factors and their Influence on Newborns Allergy risk) birth cohort was assessed for Eo/B progenitor cells (Eo/B CFU) using standardized and validated methylcellulose assays. Frozen peripheral blood mononuclear cells (PBMC) were cultured in the presence of IL-3, IL-5 or GM-CSF, and Eo/B CFUs enumerated. Clinical outcomes and exposure to environmental tobacco smoke (ETS) were documented by standardized questionnaires, and indoor volatile organic compound (VOC) concentrations were assessed by passive sampling. Children with skin manifestations (atopic dermatitis or cradle cap) within the first year of life had higher numbers of circulating IL-3-, IL-5- or GM-CSF-stimulated Eo/B CFUs (P<0.05) at 1year. In children with cradle cap, a positive correlation was found between Eo/B CFUs and exposure to ETS-related VOCs during pregnancy or at 1year of age (P<0.05). This is the first demonstration that environmental exposures are positively associated with levels of circulating Eo/B progenitors. The recruitment and differentiation of Eo/B progenitors in response to environmental triggers may play a role in the development of skin manifestations during the first year of life.

Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of Toll-like Receptor 4 (TLR4), Dendritic Cell-specific Intercellular Adhesion Molecule 3 (ICAM-3)-grabbing Nonintegrin (DC-SIGN), and β2 Integrins

Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved β2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.

Chemokine Receptor CCR5 Mediates AlloImmune Responses in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematologic malignancies. However graft-versus-host disease (GVHD) is a major limiting factor for a successful patient outcome. GVHD is a result of alloimmune responses of donor T lymphocytes attacking the recipient's cells and tissues. Chemokine receptor CCR5 plays a role in solid organ allograft rejection and mediates murine GVHD pathogenesis. Herein, we report that infiltrating lymphocytes in the skin of human acute GVHD (aGVHD) samples are predominantly CCR5(+) T cells. In addition, we characterized the features of the CCR5 expression on alloreactive T lymphocytes. We found that the CCR5(+) population exhibits the characteristics of the activated effector T cell phenotype. CCR5 expression is upregulated upon allogenic stimulation, and CCR5(+) cells are proliferating with coexpression of T cell activation markers. Furthermore, the activated T cells producing inflammatory cytokine tumor necrosis factor (TNF)alpha, interleukin (IL)-2, or interferon (IFN)-gamma, are positive for CCR5. Thus, CCR5 is a marker for GVHD effector cells and CCR5(+) T cells are active participants in the pathogenesis of human aGVHD.

Changes in T-cell subsets in HIV–HCV-coinfected patients during pegylated interferon-α2a plus ribavirin treatment

We evaluated the effect of different doses of pegylated interferon (PEG-IFN)-alpha2a/ribavirin (RBV) on several T-cell activation markers in HIV-HCV-coinfected patients and their relationship with changes in plasma HCV RNA. Frozen peripheral blood mononuclear cells (PBMCs) from 22 patients receiving two different PEG-IFN-alpha2a schedules were analysed by six-colour flow cytometry. Cell-surface expression of CD38 was quantified. HIV and HCV viral loads, as well as absolute CD4+ and CD8+ T-cell counts, were recorded during the follow up (72 weeks). PEG-IFN-alpha2a/RBV treatment decreased the absolute numbers of CD8+ and CD4+ T-cells. The decrease in CD8+ T-cells was more pronounced, resulting in increased percentages of CD4+ T-cells. Percentages of naive/memory CD4+ T-cell subsets remained unchanged, although the percentage of CD38+CD45RO+ cells significantly increased. By contrast, the CD8+ T-cell compartment significantly reduced the percentage of CD45RO+ cells and HLA-DR+ cells, whereas the percentage of CD38 expressing cells was increased because of a significant increase in cell-surface CD38 expression. Changes in CD8+ T-cells were similar for both PEG-IFN-alpha2a/RBV doses, but high doses induced more severe perturbations in CD4+ T-cells. All changes returned to baseline levels after treatment cessation and, except for the loss of naive CD4+ T-cells, were not associated with virological response. Transient lymphopaenia induced by PEG-IFN-alpha2a/RBV differentially affects T-cell subsets. Activated HLA-DR+ and CD45RO+ cells were selectively reduced in peripheral blood, whereas CD38 expression was up-regulated mainly in memory cells. Increasing PEG-IFN-alpha2a/RBV doses mainly affect CD4+ T-cells but failed to modify clinical outcome.

Long-term increase of CD4 + central memory cells in HIV-1-infected individuals by therapeutic HIV-1 rgp160 immunization

Objective To evaluate functional potential and phenotypic markers in HIV-1-infected patients immunized with HIV-1 rgp160. Methods We assessed changes in T-cell phenotype and immune function in 12 HIV-1-infected individuals that were part of a therapeutic vaccine study from 1992 to 1995 [Sandstrom E, Wahren B. Therapeutic immunisation with recombinant gp160 in HIV-1 infection: a randomised double-blind placebo-controlled trial. Nordic VAC-04 Study Group. Lancet 1999;353(9166):1735–42]. The patients received 160 μg HIV-1 rgp160 or placebo i.m. at baseline (day 0), and months 1, 2, 3, 4, 6, and thereafter every 3 months. Frozen peripheral blood mononuclear cells (PBMC) were retrieved from time points 0, 9, 12 and 24 months for phenotypic analysis utilizing flow cytometry. Results Up-regulation of immune activation markers HLA-DR and CD38 was observed at baseline and throughout the monitoring period on both CD4 + and CD8 + T cells in all patients, reflecting immune activation due to persistent high viral load. Further enhanced expression of activation markers was observed over time in the vaccine group, but not the placebo group. We also observed a consistent long-term increase of the CD4 + central memory population (CD3 +CD4 +CD45RA −CCR7 +) in the vaccinated group. Conclusions Administration of eight doses of rgp160 in a year appeared to partially reverse some of the defects exerted by HIV-1 on the immune system. A combination of vaccination with effective antiretroviral therapy (ART) may thus represent an immunotherapeutic intervention for treatment of chronic HIV-1 infection. The improvement of a HIV-1-specific central memory population and HIV-1 antigen-specific CD4 + lymphoproliferative responses may have contributed to the short-term improved survival reported in the vaccinated group.

Multicolor flow cytometric analysis of CD4+ T-cells for CD25 and Foxp3 expression in human Prostate Cancer (50.21)

Abstract CD4+CD25+ T regulatory cells (Treg) are postulated to regulate immune responses against self-antigen, infectious agents, tumor antigens and transplantation antigens. The objective of the study is to examine whether PSA146–154 peptide vaccination impacts the frequency and/or phenotype of Treg in peripheral blood mononuclear cells (PBMC) of patients with prostate cancer (CaP). Frozen PBMC were rapidly thawed, enumerated, checked for viability and directly stained with fluorescent labeled antibodies to CD3, CD4, CD8, CD25 and Foxp3 and analyzed on a flow cytometer. Based on morphology, gates were set to include, (a) whole PBMC population but exclude debris and clumps or, (b) predominantly large lymphocyte population and then back gated on CD3+CD4+ T-cells. Quadrant analysis demonstrated the presence of 1–5% of CD4+CD25+Foxp3−, less than 1% of CD4+CD25−Foxp3+ and CD4+CD25+Foxp3+ cells in the whole PBMC population. While, 10–44% of CD4+CD25−Foxp3+, 1–2% of CD4+CD25+Foxp3−, 1–3% of CD4+CD25+Foxp3+ cells were observed in the large lymphocyte population. However, there was no significant difference between before and post vaccine samples. CD4+CD25−Foxp3+ cells may represent a distinct T-cell subset and warrants further investigation. Funding: The Illinois Department of Public Health (IDPH# 4328301)