Characterization of Dendritic Cell-Specific Genes p275 and p306.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3842-3842
Author(s):  
Ingo Hilgendorf ◽  
Daniel Kurz ◽  
Anita Bringmann ◽  
Lothar Kanz ◽  
Frank Grünebach ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Splice Variant ◽  
Splice Variants ◽  
Polyclonal Antibodies ◽  
Antigen Presenting Cells ◽  
Poly I:C ◽  
Blood Monocytes ◽  
Splice Form ◽  
Antigen Presenting

Abstract Dendritic cells play an inimitable role in the functioning immune system as they are the most potent antigen presenting cells being able to prime naive T-cells. Their characteristic properties that enable them to take up antigens and present them to leukocytes are due to an expression of specific genes and thus specific proteins that are unique to this subset of antigen-presenting cells. Using a substractive cDNA library based on suppression hybridization between DC cDNA and the reference monocyte cDNA, we identified in DC two differentially expressed genes p275 and p306. p275 codes for a membrane protein and represents a splice isoform of the transport protein NAT-1. The predicted structure of protein p306 is globular, suggesting that the protein is either intracellular or secreted. The expression of both genes was confirmed by RT-PCR using cDNA isolated from peripheral blood monocytes and DC, generated in vitro from monocytes or CD34+ progenitor cells. To further analyze the protein expression polyclonal antibodies were generated by immunization with synthetic peptides deduced from the identified sequences. Interestingly, inhibition of DC differentiation using IL-10 or STI571 (Imatinib) resulted in an impaired expression of both proteins. Utilizing specific primers for two recently described splice variants of p306 we identified a new splice form expressed in DC. While the gene of p306 contains eight exons, splice variant 1 consists of the exons 1,2,4,5,6, and 7 and splice variant 2 contains the exons 1,2,3,4,5,6, a shortened exon 7, and exon 8. The new identified splice form includes the exons 1–7. However, as the open reading frame starts in exon 4, the expressed protein is identical with the one corresponding to splice variant 1. Analyzing different DC populations in peripheral blood we show that p306 is expressed in plasmacytoid, but not myeloid DC. Interestingly, the activation of DC with Toll-like receptor ligands (TLRL) Pam3Cys (TLR2L), Poly I:C (TLR3L), LPS (TLR4L) and R848 (TLR7L) has no influence on the expression of p306. Although the functions of p275 and p306 in DC have yet to be determined, both genes play a role in DC differentiation and are found in different hematopoietic cell populations. Especially p306 might be an interesting marker of plasmacytoid DC as the predicted protein does not resemble any known protein structure.

scholarly journals Impairments of Antigen-Presenting Cells in Pulmonary Tuberculosis

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Ludmila V. Sakhno ◽  
Ekaterina Ya. Shevela ◽  
Marina A. Tikhonova ◽  
Sergey D. Nikonov ◽  
Alexandr A. Ostanin ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Pulmonary Tuberculosis ◽  
Peripheral Blood ◽  
Antigen Presenting Cells ◽  
T Cell Response ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Gm Csf ◽  
Antigen Presenting

The phenotype and functional properties of antigen-presenting cells (APC), that is, circulating monocytes and generatedin vitromacrophages and dendritic cells, were investigated in the patients with pulmonary tuberculosis (TB) differing in lymphocyte reactivity toM. tuberculosisantigens (PPD-reactive versus PPD-anergic patients). We revealed the distinct impairments in patient APC functions. For example, the monocyte dysfunctions were displayed by low CD86 and HLA-DR expression, 2-fold increase in CD14+CD16+expression, the high numbers of IL-10-producing cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The macrophages which werein vitrogenerated from peripheral blood monocytes under GM-CSF were characterized by Th1/Th2-balance shifting (downproduction of IFN-γcoupled with upproduction of IL-10) and by reducing of allostimulatory activity in mixed lymphocyte culture. The dendritic cells (generatedin vitrofrom peripheral blood monocytes upon GM-CSF + IFN-α) were characterized by impaired maturation/activation, a lower level of IFN-γproduction in conjunction with an enhanced capacity to produce IL-10 and IL-6, and a profound reduction of allostimulatory activity. The APC dysfunctions were found to be most prominent in PPD-anergic patients. The possible role of APC impairments in reducing the antigen-specific T-cell response toM. tuberculosiswas discussed.

INTERDEPENDENCE BETWEEN THE PHENOTYPE OF DENDRITIC CELLS AND AMOUNTS OF BLOOD PROINFLAMMATORY MONOCYTES IN PATIENTS WITH KIDNEY CANCER

2019 ◽  
Vol 21 (4) ◽  
pp. 689-702
Author(s):  
A. A. Savchenko ◽  
A. G. Borisov ◽  
I. V. Kudryavtsev ◽  
A. V. Moshev
Keyword(s):  
Dendritic Cells ◽  
Kidney Cancer ◽  
Peripheral Blood ◽  
Receptor Expression ◽  
Autologous Serum ◽  
Autologous Tumor ◽  
Blood Monocytes ◽  
Inflammatory Monocytes ◽  
Antigen Presenting

The aim of the study was to investigate an interdependence between the phenotype of dendritic cells (DC) differentiated from monocytes and the number of pro-inflammatory monocytes in peripheral blood of patients with kidney cancer (KC). The study involved 28 patients at the age of 40-55 years suffering with KC (Т3N0М0, clear cell type) before surgical treatment. The diagnosis was verified histologically. 31 healthy agematched persons were examined as a control group. Mononuclear cells were isolated from heparinized venous blood by centrifugation in a Histopaque®-1077 density gradient followed by plastic adsorption in RPMI 1640 medium supplied with 10% autologous serum. Immature DCs (iDCs) were generated from blood monocytes by culturing for 5 days with GM-CSF and IFNα. Activation of DCs (mDCs) was induced by incubation with the tumor cell lysate and TNFα, followed by incubation for 48 hours. A tumor fragment was used to prepare the lysate of autologous tumor cells. Phenotyping of blood monocytes and DC at various maturation stages was performed by flow cytometry. The numbers of CD14+CD16+ monocytes in peripheral blood of KC patients were decreased (up to 42% of the total monocyte level) against the control ranges. In this regard, the analysis of the dependence between the phenotype of DCs differentiated from monocytes and the number of pro-inflammatory blood monocytes was carried out by comparing the groups with a high content of pro-inflammatory monocytes in the blood in KC patients (> 42%, near-control range) and low content (resp., < 42%). We have found that the contents of tolerogenic iDC in cell culture are increased in KC patients with low amounts of pro-inflammatory monocytes in blood (< 42%). A relatively increased expression of antigen-presenting and co-stimulatory molecules proved to be the specific feature of iDC phenotype in patients with high contents (> 42%) of proinflammatory monocytes in blood. The phenotype of dendritic cells in KC patients with different content of proinflammatory monocytes during maturation/activation showed more differences. In the patients with low levels of pro-inflammatory monocytes, the cell pool of in vitro maturing DCs was characterized by low level of CD86 and HLA-DR receptor expression, thus reflecting a weak co-stimulating and antigen-presenting activity. In the patients with high levels of pro-inflammatory monocytes in blood, the in vitro activated DCs showed higher level of functional activity using the above markers. The revealed differences in the DC phenotype and interrelations with amounts of blood monocyte subpopulations in KC patients may presume the programmed cell differentiation mechanisms depending on the microenvironment, under pathogenic conditions (i.e., in presence of malignant tumor growth).

scholarly journals Generation of Feline Dendritic Cells Derived from Peripheral Blood Monocytes for In Vivo Use

2005 ◽  
Vol 12 (10) ◽  
pp. 1202-1208 ◽  
Author(s):  
Giulia Freer ◽  
Donatella Matteucci ◽  
Paola Mazzetti ◽  
Leonia Bozzacco ◽  
Mauro Bendinelli
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Peripheral Blood ◽  
Poly I:C ◽  
Interleukin 4 ◽  
Blood Monocytes ◽  
Autologous Plasma ◽  
Peripheral Blood Monocytes

ABSTRACT Dendritic cells (DCs) are professional antigen-presenting cells that can prime T cells and polarize the cellular immune response. Because Th1-type immune responses have been connected to success in combating viral infection, a promising therapeutic application of DCs would be their differentiation in vitro and injection back into the host to boost an immune response in infected animals. This study was aimed both at developing a protocol to cultivate feline DCs in the absence of exogenous proteins for their use in vivo and at investigating what might be the most appropriate stimulus to induce their maturation in vitro and finding correlates of maturation. We generated DCs from peripheral blood monocytes in the presence of feline interleukin-4 and granulocyte-macrophage colony stimulating factor, and after 5 days their maturation was induced with either lipopolysaccharide, human recombinant tumor necrosis factor alpha, poly(I:C), or activated feline platelets. After 48 h, their CD14, CD1a, major histocompatibility complex class II, and B7.1 surface expression was analyzed in parallel with their ability to uptake antigen or prime a mixed leukocyte reaction. The results presented show that feline DCs cultured in autologous plasma differentiate and are able to mature in the presence of stimuli similar to the ones currently used for other species. The present work sets the grounds for future use of DCs obtained by the protocol described for in vivo vaccination and immunotherapy of feline immunodeficiency virus-infected cats.

scholarly journals Development of Stable Chimeric IL-15 for Trans-Presentation by the Antigen Presenting Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Manoj Patidar ◽  
Naveen Yadav ◽  
Sarat K. Dalai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Half Life ◽  
Therapeutic Potential ◽  
Chimeric Protein ◽  
Antigen Presenting Cells ◽  
T Cell Response ◽  
Antigen Presenting

IL-15 is one of the important biologics considered for vaccine adjuvant and treatment of cancer. However, a short half-life and poor bioavailability limit its therapeutic potential. Herein, we have structured IL-15 into a chimeric protein to improve its half-life enabling greater bioavailability for longer periods. We have covalently linked IL-15 with IgG2 base to make the IL-15 a stable chimeric protein, which also increased its serum half-life by 40 fold. The dimeric structure of this kind of IgG based biologics has greater stability, resistance to proteolytic cleavage, and less frequent dosing schedule with minimum dosage for achieving the desired response compared to that of their monomeric forms. The structured chimeric IL-15 naturally forms a dimer, and retains its affinity for binding to its receptor, IL-15Rβ. Moreover, with the focused action of the structured chimeric IL-15, antigen-presenting cells (APC) would transpresent chimeric IL-15 along with antigen to the T cell, that will help the generation of quantitatively and qualitatively better antigen-specific memory T cells. In vitro and in vivo studies demonstrate the biological activity of chimeric IL-15 with respect to its ability to induce IL-15 signaling and modulating CD8+ T cell response in favor of memory generation. Thus, a longer half-life, dimeric nature, and anticipated focused transpresentation by APCs to the T cells will make chimeric IL-15 a super-agonist for memory CD8+ T cell responses.

scholarly journals Complex CSF3R Protein Isoform Interactions Dictate Cytokine Responsiveness

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-35
Author(s):  
Sara L. Seegers ◽  
Amanda Lance ◽  
Lawrence J Druhan ◽  
Belinda R Avalos
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Splice Variant ◽  
Splice Variants ◽  
Polyclonal Antibodies ◽  
Human Cells ◽  
Dependent Manner ◽  
Epitope Tag ◽  
Proliferative Signaling ◽  
Primary Human Cells

CSF3R, the receptors for granulocyte colony stimulating factor, is a critical regulator of neutrophil production. Multiple CSF3R mRNA transcripts have been identified and are annotated in Genbank. The expression and function of the different CSF3R proteins have not been fully elucidated. We generated antibodies specific for two of the identified and annotated isoforms, V3 and V4. CSF3R-V4 is a truncated variant of V1 with a unique C-terminal 34 amino acids and this variant confers enhanced growth signals. Changes in the ratio of V1:V4 isoforms have been implicated in chemotherapy resistance and relapse of AML. CSF3R-V3 is a variant of V1 with a 27 amino acid insertion between two conserved domains in the cytoplasmic portion of the receptor involved in JAK/STAT activation, termed the box 1 and box 2. CSF3R-V3 produces reduced proliferative signaling in response to G-CSF. When V3 is co-expressed with V1, proliferative signaling is reduced in a concentration dependent manner. In order to generate custom rabbit polyclonal antibodies specific for CSF3R-V3 and CSF3R-V4 we used either a peptide that corresponds to a unique amino acid sequence present only in CSF3R-V3 or a peptide specific for a portion of the C-terminal amino acid sequence unique to the CSF3R-V4 isoform conjugated to an immunogenic carrier protein. These immunogens both produced robust immune responses, and the polyclonal antibodies were subsequently purified from bulk sera. Immunoblot analysis of lysates from Ba/F3 cells expressing CSF3R-V1 (V1), CSF3R-V3 (V3), or CSF3R-V4 (V4) demonstrated that both the custom generated anti-CSF3R-V3 and anti-CSF3R-V4 antibodies were very specific, recognizing only the appropriate CSF3R receptor isoform. All three CSF3R splice variants are recognized by commercially available anti-CSF3R (clone LMM741 to CD114), while the anti-CSF3R-V4 custom antibody and the custom anti-CSF3R-V3 antibody recognizes only the CSF3R-V4 and CSF3R-V3 isoforms, respectively. We next sought to detect the CSF3R receptor isoforms in primary human cells. Using our custom antibodies, we detected for the first time, both the CSF3R-V3 and CSF3R-V4 receptor forms in primary neutrophils isolated from healthy donors. Each of the CSF3R isoforms produce unique signaling, and we hypothesized that the observed differences in G-CSF-dependent signaling is produced by the expression level of each receptor isoform via both homodimerization and by heterodimerization of the receptor splice variant proteins. To investigate the potential for heterodimerization of the CSF3R-V1 with the V3 and V4 isoforms, we generated a CSF3R-V1 with a c-terminal epitope tag and co-expressed this construct with both CSF3R-V3 or CSF3R-V4. Immunoprecipitation with an antibody to the epitope tag (recognizing the V1 variant) followed by immunoblotting with the custom anti-V3 or anti-V4 antibodies demonstrated that both CSF3R-V3 and CSF3R-V4 co-immunoprecipitated with CSF3R-V1, in agreement with our hypothesis that the splice variants form receptor heterodimers. Of note, the CSF3R receptor heterodimers are detected even in the absence of G-CSF, thus demonstrating that CSF3R exist as a preformed receptor dimer in an inactive state. In conclusion, we have generated antibodies that specifically detect the CSF3R-V3 and the CSF3R-V4 receptor proteins. These are the first studies to demonstrate the expression of the CSF3R splice variants at the protein level, in both cell lines and primary human cells. In addition, these are the first studies to demonstrate the formation of heterodimers of the CSF3R splice variants, providing a mechanism for the observed alteration in ligand-dependent signaling produced under conditions of altered splice variant expression. Disclosures Avalos: Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia.

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