Stefanie Slezak

CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects

BackgroundAdoptive immune and vaccine therapies have been used to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic progenitor cell transplants, but the nature of T cell responses to CMV have not been completely characterized.MethodsPeptide pools and individual peptides derived from the immune-dominant CMV proteins pp65 and IE-1 and antigen-specific, cytokine flow cytometry were used to characterize the prevalence and frequency of CD4+ and CD8+ memory T cells in 20 healthy CMV-seropositive subjects.ResultsCD8+ T cell responses to pp65 were detected in 35% of subjects and to IE-1 in 40% of subjects. CD4+ T cell responses to pp65 were detected in 50% of subjects, but none were detected to IE-1. Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens. One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes. The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides.ConclusionSince naturally occurring CD4+ and CD8+ T cell responses to pp65 were detectable in many subjects, but only CD8+ T cell responses to IE-1 were detected, pp65 may be better than IE-1 for use in vaccine and adoptive immune therapies.

Evaluation of 3 Clinical Dendritic Cell Maturation Protocols Containing Lipopolysaccharide and Interferon-γ

Dendritic cells (DCs) are important adjuvants for cancer vaccines. Immature dendritic cells (iDCs) are often produced by the stimulation of peripheral blood monocytes with interleukin (IL)-4 and granulocyte macrophage-colony stimulating factor. For many applications iDCs are treated with cytokines or inflammatory signals to produce mature DCs (mDCs). iDCs are often treated ex vivo with lipopolysaccharide (LPS) and interferon (IFN)-γ to produce mDCs for clinical therapy. The purpose of this study was to determine if the DC maturation cocktail LPS plus IFN-γ could be improved by the addition of 2 other DC maturation agents IL-1β and tumor necrosis factor (TNF)-α. Peripheral blood mononuclear cells were collected from 6 healthy subjects. Monocytes were isolated from the peripheral blood mononuclear cell concentrates by elutriation and were incubated for 3 days with granulocyte macrophage-colony stimulating factor and IL-4 to produce iDCs. iDCs from each subject were divided into 3 and were incubated for 24 hours with LPS plus IFN-γ; LPS, IFN-γ, plus IL-1β; or LPS, IFN-γ, IL-1β, plus TNF-α to produce mDCs. The DCs were compared by measuring the expression of costimulator and antigen presenting molecules (CD80, CD83, CD86, and human leukocyte antigen-DR) by flow cytometry, cytokine production (IL-12p70 and IL-10) by enzyme-linked immunosorbent assay and global gene expression using an oligonucleotide microarray. There were no differences in the expression of costimulatory molecules, human leukocyte antigen-DR and CCR7 and production of IL-12p70 among the mDCs produced with the 3 cocktails. Global gene expression analysis found that the expression of 9576 genes differed between the iDCs and mDCs, but the expression of only 13 differed among the 3 different groups of mDCs. There was no benefit of adding IL-1β and TNF-α to LPS and IFN-γ to produce mDCs.

Comparison of proteomic profiles of serum, plasma, and modified media supplements used for cell culture and expansion

BackgroundThe culture and expansion of human cells for clinical use requires the presence of human serum or plasma in culture media. Although these supplements have been extensively characterized in their chemical composition, only recently it has been possible to provide by high throughput protein analysis, a comprehensive profile of the soluble factors contributing to cell survival. This study analyzed and compared the presence of 100 proteins including chemokines, cytokines and soluble factors in six different types of media supplements: serum, plasma, recalcified plasma, heat inactivated serum, heat inactivated plasma and heat inactivated recalcified plasma.MethodsSerum, plasma, recalcified plasma, and heat inactivated supplements were prepared from ten healthy subjects. The levels of 100 soluble factors were measured in each sample using a multiplexed ELISA assay and compared by Eisen hierarchical clustering analysis.ResultsA comparison of serum and plasma levels of soluble factors found that 2 were greater in plasma but 18 factors were greater in serum including 11 chemokines. The levels of only four factors differed between recalcified plasma and plasma. Heat inactivation had the greatest effect on soluble factors. Supervised Eisen hierarchical clustering indicated that the differences between heat inactivated supplements and those that were not were greater than the differences within these two groups. The levels of 36 factors differed between heat inactivated plasma and plasma. Thirty one of these factors had a lower concentration in heat inactivated plasma including 12 chemokines, 4 growth factors, 4 matrix metalloproteases, and 3 adhesion molecules. Heat inactivated decalcified plasma is often used in place of heat inactivated serum and the levels of 19 soluble factors differed between these two supplements.ConclusionOur report provides a comprehensive protein profile of serum, plasma recalcified plasma, and heat inactivated supplements. This profile represents a qualitative and quantitative database that can aid in the selection of the appropriate blood derived supplement for human cell cultures with special requirements.

Gene and microRNA analysis of neutrophils from patients with polycythemia vera and essential thrombocytosis: down-regulation of micro RNA-1 and -133a

BackgroundSince the V617F mutation in JAK2 may not be the initiating event in myeloprofilerative disorders (MPDs) we compared molecular changes in neutrophils from patients with polycythemia vera (PV) and essential thrombocythosis (ET), to neutrophils stimulated by G-CSF administration and to normal unstimulated neutrophilsMethodsA gene expression oligonucleotide microarray with more than 35,000 probes and a microRNA (miR) expression array with 827 probes were used to assess neutrophils from 6 MPD patients; 4 with PV and 2 with ET, 5 healthy subjects and 6 healthy subjects given G-CSF. In addition, neutrophil antigen expression was analyzed by flow cytometry and 64 serum protein levels were analyzed by ELISA.ResultsGene expression profiles of neutrophils from the MPD patients were similar but distinct from those of healthy subjects, either unstimulated or G-CSF-mobilized. The differentially expressed genes in MPD neutrophils were more likely to be in pathways involved with inflammation while those of G-CSF-mobilized neutrophils were more likely to belong to metabolic pathways. In MPD neutrophils the expression of CCR1 was increased and that of several NF-κB pathway genes were decreased. MicroRNA miR-133a and miR-1 in MPD neutrophils were down-regulated the most. Levels of 11 serum proteins were increased in MPD patients including MMP-10, MMP-13, VCAM, P-selectin, PDGF-BB and a CCR1 ligand, MIP-1α.ConclusionThese studies showed differential expression of genes particularly involved in inflammatory pathways including the NF-κB pathway and down-regulation of miR-133a and miR-1. These two microRNAs have been previous associated with certain cancers as well as the regulation of hyperthrophy of cardiac and skeletal muscle cells. These changes may contribute to the clinical manifestations of the MPDs.

Evaluation of gene expression profiles of immature dendritic cells prepared from peripheral blood mononuclear cells

BACKGROUND: Dendritic cells (DCs) generated ex vivo from peripheral blood monocytes or mobilized CD34+ cells and intended for clinical immunotherapy are typically characterized by morphologic, phenotypic, and functional assays. Assay results are highly dependent on conditions used to prepare the cells, so there is no standard assay battery for clinical DC products. This study evaluated gene expression profiling for characterization of immature DCs prepared from monocytes that had been elutriated from normal donor peripheral blood mononuclear cells (PBMNCs) immediately after collection or after storage at 4°C for 48 hours.

GM-CSF/IL-3/IL-5 receptor common β chain (CD131) expression as a biomarker of antigen-stimulated CD8 + T cells

BackgroundUpon Ag-activation cytotoxic T cells (CTLs) produce IFN-γ GM-CSF and TNF-α, which deliver simultaneously pro-apoptotic and pro-inflammatory signals to the surrounding microenvironment. Whether this secretion affects in an autocrine loop the CTLs themselves is unknown.MethodsHere, we compared the transcriptional profile of Ag-activated, Flu-specific CTL stimulated with the FLU M1:58-66 peptide to that of convivial CTLs expanded in vitro in the same culture. PBMCs from 6 HLA-A*0201 expressing donors were expanded for 7 days in culture following Flu M1:58-66 stimulation in the presence of 300 IU/ml of interleukin-2 and than sorted by high speed sorting to high purity CD8+ expressing T cells gated according to FluM1:58-66 tetrameric human leukocyte antigen complexes expression.ResultsAg-activated CTLs displayed higher levels of IFN-γ, GM-CSF (CSF2) and GM-CSF/IL-3/IL-5 receptor common β- chain (CD131) but lacked completely expression of IFN-γ receptor-II and IFN-stimulated genes (ISGs). This observation suggested that Ag-activated CTLs in preparation for the release of IFN-γ and GM-CSF shield themselves from the potentially apoptotic effects of the former entrusting their survival to GM-SCF. In vitro phenotyping confirmed the selective surface expression of CD131 by Ag-activated CTLs and their increased proliferation upon exogenous administration of GM-CSF.ConclusionThe selective responsiveness of Ag-activated CTLs to GM-CSF may provide an alternative explanation to the usefulness of this chemokine as an adjuvant for T cell aimed vaccines. Moreover, the selective expression of CD131 by Ag-activated CTLs proposes CD131 as a novel biomarker of Ag-dependent CTL activation.

Analysis of vaccine-induced T cells in humans with cancer.

Over the past several years, progress in the field of tumor immunology has lead to advances in active immunotherapy and vaccination as a means ofeliciting tumor-specific immune responses to mediate tumor regression and clearance. Developing vaccines targeted against cancer became an important focus as a therapy following the success of viral vaccines in preventing infection and disease. In humans with cancer, similar to viral infections, the host immune system is capable of recognizing antigens expressed on tumor cells. This similarity allows the immunological framework of the viral vaccine to be adapted to the cancer setting in hopes of enhancing human T-cell reactivity against tumor. It is generally believed that a requirement for tumor destruction to occur is the induction of sufficient levels of immune cells with high avidity for recognition of tumor antigens. Moreover, the cells must be targeted to the tumor site and be capable of infiltrating tumor stroma.2 Several tumor-associated antigens (TAA) have been identified in the melanoma model which has allowed for immunization trials to evaluate therapeutic potential of tumor-specific T-cell induction. Some clinical trials reported limited success ofT-cell mediated tumor rejection, reportingpartial or complete regression in 10 to 30% of patients. Although tumor regression was not observed following active immunization in vivo, ex vivo assays evaluating TAA-specific T cells demonstrated tumor recognition and subsequent T-cell activation suggesting that tumor-specific T-cell induction indeed occurs but alone is not adequate to induce tumor regression. Recently, the usefulness and success of active-specific immunization (ASI) against TAAs as a means ofeliciting a tumor-specific immune response leading to tumor regression and clearance has been a topic of debate and discussion.

Another example of a KEL1 variant red cell phenotype due to a threonine to serine change at position 193 of Kell glycoprotein.

BACKGROUND: Healthy subjects whose red blood cells (RBCs) react variably with anti‐KEL1, but strongly express other Kell blood group antigens, have been described and called KEL1 variant. A 53‐year‐old Caucasian blood donor was identified whose RBCs reacted with three monoclonal and two polyclonal anti‐KEL1 and did not react with two monoclonal and one polyclonal anti‐KEL1. The molecular basis of this phenotype was investigated.