Autologous Peripheral Blood Monocytes Research Articles

Intrauterine CRH-treated PBMC in repeated implantation failure.

The intrauterine administration of activated autologous peripheral blood monocytes (PBMC) prior to embryo transfer seems to improve reproductive outcomes in women with repeated implantation failure (RIF). We have previously shown that the intrauterine administration of PBMC treated with corticotropin-releasing hormone (CRH) prior to blastocyst transfer (day 5) improves significantly the clinical pregnancy rate of women with RIF. In the present crossover pilot study, we have investigated whether CRH-PBMC treatment could be of benefit in case of fresh early cleavage stage embryo transfer (day 3) in women with RIF. Twenty-six (n=26) women with at least three previous failed IVF attempts and no history of clinical pregnancy in the past were recruited in this study. Ovarian stimulation was performed following either the long or the short protocol. PBMC were collected during the oocyte retrieval, were treated with CRH, and transferred in the uterine cavity 2days later. Good quality cleavage stage embryos were transferred at day 3, following oocyte retrieval. Following the intrauterine administration of CRH-treated autologous PBMC, 15/26 clinical pregnancies occurred (57.69%). Compared to the null result of the same women prior to recruitment, this observation was considered significant (P<10-2 ). Our findings further support the role of the intrauterine administration of CRH-treated PBMC as an effective approach when transferring cleavage stage embryos in women with RIF. Prospective randomized studies are needed to clarify whether such intervention could be of benefit in clinical practice.

Macrophages from the upper and lower human respiratory tract are metabolically distinct

The function and cell surface phenotype of lung macrophages vary within the respiratory tract. Alterations in the bioenergetic profile of macrophages may also be influenced by their location within the respiratory tract. This study sought to characterize the bioenergetic profile of macrophages sampled from different locations within the respiratory tract at baseline and in response to ex vivo xenobiotic challenge. Surface macrophages recovered from healthy volunteers by induced sputum and by bronchial and bronchoalveolar lavage were profiled using extracellular flux analyses. Oxygen consumption and extracellular acidification rates were measured at rest and after stimulation with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), or 1,2-naphthoquinone (1,2-NQ). Oxygen consumption and extracellular acidification rates were highly correlated for all macrophage samples. Induced sputum macrophages had relatively higher oxygen consumption and extracellular acidification rates and were largely reliant on glycolysis. In contrast, bronchial fraction and bronchoalveolar macrophages depended more heavily on mitochondrial respiration. Bronchoalveolar macrophages showed elevated LPS-induced cytokine responses. Unlike their autologous peripheral blood monocytes, lung macrophages from any source did not display bioenergetic changes following LPS stimulation. The protein kinase C activator PMA did not affect mitochondrial respiration, whereas the air pollutant 1,2-NQ induced marked mitochondrial dysfunction in bronchoalveolar and bronchial fraction macrophages. The bioenergetic characteristics of macrophages from healthy individuals are dependent on their location within the respiratory tract. These findings establish a regional bioenergetic profile for macrophages from healthy human airways that serves as a reference for changes that occur in disease.

Plasma from some cancer patients inhibits adenoviral Ad5f35 vector transduction of dendritic cells

BackgroundPooled AB serum is often used as a media supplement for cell culture but it has the potential to transmit infectious diseases. To avoid this risk, we used autologous plasma as a media supplement for manufacturing dendritic cells (DCs) for cancer immunotherapy. We noticed inconsistencies in the DCs and investigated their nature and cause. MethodsAdenovirus human epidural growth factor receptor 2 (adHER2/neu) DCs for 21 patients were manufactured from autologous peripheral blood monocytes that were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 3 days, transduced with Ad5f35HER2ECTM and then treated with lipopolysaccharide and interferon (IFN)-γ for 1 day. The cells were cultured in RPMI-1640 supplemented with either 10% heat inactivated autologous or AB plasma. ResultsTwenty-eight adHER2/neu DCs were manufactured for 21 patients using autologous plasma and 68 were manufactured for 20 of those patients using AB plasma. The expression of human epidural growth factor receptor 2 (HER2/neu) was less for DCs manufactured with autologous plasma (70.3 ± 33.3% versus 86.1 ± 22.8%; P < 0.01). Manufacturing adHER2/neu DCs using monocytes from three healthy subjects and plasma from one patient with low HER2/neu expression (18%) resulted in low HER2/neu expression by all three DCs (13%, 16% and 23%). Analysis of the levels of 1322 proteins in eight plasma samples associated with low HER2/neu expression and in 12 associated with high HER2/neu expression revealed that the levels of 14 predicted HER2/neu transduction efficiency. ConclusionThe manufacture of adHER2/neu DC using autologous plasma as a media supplement resulted in inconsistent HER2/neu expression. It is likely that variability in the levels of multiple proteins in autologous plasma contributed to low HER2/neu expression.

Autoreactive T Cells From Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Specifically Recognize Glycosyl- Phosphatidyl-Inositol (GPI)

AbstractAbstract 647PNH is caused by somatic mutations in the PIG-A gene in hematopoietic stem cells leading to disruption of the synthesis of GPI, a glycolipid used for post-translational modification of proteins. PNH is classically characterized by the triad of intravascular hemolysis, severe acquired thrombophilia and multiple cytopenias. Whereas the first two components are the consequence of increased susceptibility to activated complement, due to the deficiency on red cells of GPI-linked CD55 and CD59, cytopenias are an expression of bone marrow failure. This fact, together with strong clinical evidence associating PNH with aplastic anemia, has led to the notion that a T cell-mediated attack underlies both conditions whereby in PNH the PIG-A mutant clone is able to escape the attack. Consistent with this, we previously found skewing of the spectratyping pattern of T cells, and recurrent TCR beta chain sequences. Here, using complementary assays, we tested the hypothesis that PIG-A mutant cells escape an auto-immune T cell attack targeted to the GPI molecule itself, which would be presented by CD1d.First, CD8+ T cells from PNH patients and from normal donors were co-cultured with C1R-CD1d B cells pulsed with either trypanosomal GPI (t-GPI) or with solvent. Reactive T cells producing interferon gamma (IFNG) were then enumerated by ELISPOT technology. We found that in the presence of t-GPI-pulsed C1R-CD1d cells, the frequency of reactive T cells was significantly increased (up to 5-fold) in patients on each of days 1, 7, 14 but not in controls. To confirm that T cell reactivity to GPI depended on the presence of CD1d, when similar experiments were carried out with the untransfected, CD1d-negative C1R cells, no such increase was seen. Similar results were obtained using as antigen presenting cells (APC) K562 cells and appropriate derivatives.Next, we used for antigen presentation, in addition to the C1R-CD1d cell line, a derivative that lacks the ability to synthesize GPI (GPI- C1R-CD1d). In these experiments, reactive (IFNG-producing) T cells were counted by intracellular staining and flow-cytometry:in PNH patients, but not in controls, we found a 3-fold increase in the frequency of reactive CD8+ T cells when the APC were GPI+ C1R-CD1d compared to when they were GPI- C1R-CD1d. This increase was not seen when APC were the C1R cells. By contrast, with GPI- C1R-CD1d the increase was restored by the addition of exogenous human GPI (h-GPI).Next, we tested the reactivity of T cells from 11 PNH patients using as APC mature dendritic cells (DC) obtained from autologous peripheral blood monocytes. By selecting CD14-neg cells – that are the majority in PNH patients - we took advantage of the fact that in this way we had APC that were naturally GPI negative. In the absence of added GPI we could not demonstrate reactive T cells; however, in 7 out of 11 patients there was a significant increment when the DC were loaded with h-GPI (p =0,0149; Mann-Whitney test).Since CD1d is required for development and function of iNKT cells, immunoregulatory, glycolipid-specific T cells characterized by an invariant TCRValpha24Jalpha18 chain, we tested the hypothesis that CD1d-restricted, GPI-specific T cells might also possess an invariant TCR alpha chain. Global TCR alpha chain repertoire analysis of flow-sorted dimer CD1d/GPI+ T cells identified a novel TCRValpha21Jalpha31 invariant chain (9/9 clones) in 1 out of 3 patients tested. Further analysis of the TCRValpha21 repertoire of patients and controls by next generation sequencing (GS Junior, Roche) revealed that the invariant TCRValpha21Jalpha31 chain was present but not expanded in 5/8 controls (frequency<0.5% of TCRValpha21-Calpha T cell repertoire); by contrast, it was expanded in 6/11 patients (median frequency: 1.86%, range: 1.05–75% of TCRValpha21-Calpha T cell repertoire).In conclusion, we provide the first direct evidence that GPI-specific, CD1d-restricted T cells, enriched in a novel invariant TCR alpha chain sequence, are increased in patients with PNH but not normal controls. Disclosures:No relevant conflicts of interest to declare.

The TLR7/8 Ligand R848 Is Superior In Generation of Monocyte Derived Dendritic Cells From AML Patients for the Induction of Potent T and NK Cell Immune Responses

AbstractAbstract 2195 Introduction:Therapeutic vaccination with dendritic cells (DC) is currently considered as an investigational therapy in acute myeloid leukemia (AML) for eradication of minimal residual disease (MRD). Dendritic cells derived from autologous peripheral blood monocytes have been tested as cellular adjuvants for therapeutic vaccination of malignancies and proven feasibility and safety, but overall clinical response rates remain very low. The vast majority of DCs used for clinical trials were differentiated with a standard maturation cocktail composed of the cytokines TNF-a, IL-1b, IL-6 and PGE2 leading to DCs unable to secrete biologically active IL-12. This cytokine is fervently desired because of its leading role in promoting T helper 1 cell polarization and therefore fostering the appropriate adaptive immune responses needed to combat minimal residual disease. Cocktails containing synthetic Toll-like receptors (TLR) agonists emerged as an attractive alternative for the induction of DC maturation with T helper type 1 polarizing capacity. Our present investigation was designed to study the feasability of a clinical grade DC 3-day mDC generation protocol from nonleukemic monocytes of intensively pretreated AML patients with novel maturation cocktails containing different TLR-agonists in vitro and assessment of their potency to induce adaptive and innate immune responses. Material & Methods:Monocytes isolated from peripheral blood of AML patients in CR and healthy donors were differentiated into immature DC with GM-CSF and IL-4. After 48 hours DC were additionally cultured with TNF-a, IL1-b, INF-g, PGE2 and corresponding to the defined cocktail with the TLR7/8 agonists R848 (R) or CL075 (C) with or without the TLR3 agonist poly(I:C) (P) for 24 hours. mDCs were analyzed for expression of maturation surface markers, costimulatory profile, IL-12(p70)/IL-10 ratio, migratory capacity, NK cell activation and polarization of T cells. Results:No significant difference in absolute monocyte counts and percentage of DC recovery between healthy controls and AML patients in CR was found using different maturation cocktails (C, CP, R, and RP). Phenotype analysis of surface marker expression revealed no substantial differences between the different DC generation protocols used in healthy donors and AML patients in CR. The costimulatory profile assesed by the expression of two members of the B7 family, CD80 (B7.1) and CD274 (B7-H1 or PD-L1), was in healthy donors superior to AML patients, but these differences were not statistically significant. Variations were noted in the capacity of DCs derived from different donors to produce IL-12(p70) and IL-10, but importantly no significant differences between AML patients in CR and healthy controls could be observed. Interestingly, both healthy donor and AML derived DCs secrete a significantly higher proportion of IL-12(p70) with R848 containing cocktails compared to CL075. Treatment with the CP cocktails even leads to a inverse Il12/IL-10 ratio in AML patients. The high CCR7 expression was paralleled by a strong migratory capacity as well as positive chemotactic reponses to CCL19 chemokine signals. DCs matured with these novel cocktails induced potent alloresponses and strongly activated NK cells measured by upregulation of CD69 expression and IFN-g secretion. No differences beetween R848 and CL075 could be observed. Conclusion:Here we report for the first time a clinically applicable, time- and resource saving 3-day TLR-agonist containing maturation protocol for the generation of IL-12(p70) secreting DCs from AML patients in remission validated with healthy controls which allowed efficient generation, easy harvesting, stable maturation and substantial recoveries of mature DCs. Comparison of different TLR7/8 agonists showed superiority of R848 in IL-12(p70) production to CL075. We believe that these studies point the way to improved DCs that will induce better and long lasting immune responses in the vaccination against acute myelo Disclosures:No relevant conflicts of interest to declare.

Comparison of the T lymphocyte-dependent induction of angiotensin-converting enzyme and leucine aminopeptidase in cultured human monocytes

The T lymphocyte-mediated induction of angiotensin-converting enzyme (ACE) in cultured autologous peripheral blood monocytes has been proposed as a model system for the investigation of the in vivo induction of ACE in the monocyte-derived granuloma epithelioid cells of some granulomatous diseases such as sarcoidosis. The studies described here were designed to evaluate the specificity of the model system by comparing the parameters for induction of ACE with those for the induction of another monocyte metallo-ecto-peptidase, leucine aminopeptidase (LAP). The concentration of LAP in freshly isolated monocytes was 0.09 mU/10(6) monocytes (s.e.m. 0.04) and increased to a maximal value of 0.19 mU/10(6) monocytes (s.e.m. 0.32) after 3 days when monocytes were cultured alone. ACE was not detectable in freshly isolated monocytes. However, after 6 days of culture, monocytes contained 0.22 mU ACE/10(6) monocytes (s.e.m. 0.04). Comparison of the levels of ACE and LAP induced during culture of monocytes alone indicated that the induction of these two enzymes were correlated. The induction of both enzymes was further enhanced by the presence of T lymphocytes in a dose-dependent manner. At 4 x 10(6) T lymphocytes per culture, ACE levels increased to 1.81 mU/10(6) monocytes (s.e.m. 0.24) and LAP levels to 1.03 mU/10(6) monocytes (s.e.m. 0.35). The enhancement of ACE activity required autologous lymphocytes, while heterologous T lymphocytes were equally effective in inducing LAP. Comparison of the levels of ACE and LAP induced during coculture of autologous T lymphocytes and monocytes from 21 independent donors, demonstrated no correlation between the induction of ACE and LAP. These data indicate that, although T lymphocytes also enhance the induction of LAP, the underlying mechanism must differ from that of ACE induction.

Peripheral blood monocytes from the decompensated liver cirrhosis could migrate into nude mouse liver with human hepatocyte-markers expression

Adult stem cells provide a promising alternative for the treatment of decompensated liver cirrhosis. Our previous study showed that peripheral blood monocytes (PBMCs) from decompensated liver cirrhosis could differentiate into hepatocyte-like cells in vitro. We here aimed to investigate the differential potential of the PBMCs under liver environment. PBMCs were isolated from one cirrhotic patient, who was mobilized by the recombinant human granulocyte colony stimulating factor for consecutive 3 days, and then PBMCs were transplanted into nude mice by tail vein after labeled with PKH26-GL. The location of the transplanted PBMCs was identified by PKH26-GL staining and PRINS for human SRY gene. The expressions of human hepatocyte-markers were detected by immunohistochemistry, RT-PCR, and Western blot analysis. Our results demonstrated that PBMCs from decompensated liver cirrhosis could migrate into the liver of nude mice with human hepatocyte-markers expression, indicating that autologous PBMCs transplantation might be one alternative therapy for the decompensated liver cirrhosis.

Increases in 5-lipoxygenase activating protein expression account for enhanced capacity for 5-lipoxygenase metabolism that accompanies differentiation of peripheral blood monocytes into alveolar macrophages.

The capacity for 5-lipoxygenase (5-LO) metabolism of endogenous arachidonic acid (AA) is greater in alveolar macrophages (AM) than in their circulating precursors, peripheral blood monocytes (PBM); however, the ability of PBM to metabolize exogenous AA to 5-LO products is comparable to that of AM. In the present study, we examined the enzymatic mechanisms by which 5-LO metabolism of AA is altered during differentiation of PBM in the lung. Resting human AM exhibited greater steady-state levels of 5-LO (7-fold) and LTA4 hydrolase (2-fold) proteins than autologous PBM; moreover, they differed from PBM in that they contained a significant amount of 5-LO associated with the particulate fraction. Importantly, AM contained 40-fold more 5-lipoxygenase activating protein (FLAP) than did PBM, which correlated well with the relative abilities of intact AM and PBM to metabolize endogenous AA to leukotrienes. The FLAP inhibitor MK-886 was unable to block leukotriene synthesis from exogenous AA in the two cell types, despite its ability to completely inhibit 5-LO metabolism of endogenous AA. These observations indicate that, although FLAP is essential for the synthesis of leukotrienes from endogenous AA, perhaps by presenting AA to 5-LO, it is not required for 5-LO metabolism of exogenous AA. The differing roles of FLAP in 5-LO metabolism of endogenous versus exogenous AA are consistent with the conclusion that it is the markedly greater expression of FLAP, rather than of 5-LO, that is primarily responsible for the increased leukotriene synthesis from endogenous AA that accompanies PBM differentiation into AM.

Tumor necrosis factor-alpha production by alveolar macrophages in heart-lung transplant recipients.

Tumor necrosis factor-alpha (TNF alpha) is a cytokine produced by mononuclear cells that amplifies inflammation and modulates expression of Class I and Class II histocompatibility antigens. Because of these properties, this cytokine may exert a central role in both the defense and the rejection of the transplanted lung. Utilizing an ELISA technique, we measured TNF alpha in vivo and in vitro in several compartments of lung transplant recipients and in normal subjects that included serum, bronchoalveolar lavage fluid (BAL), and media conditioned by alveolar macrophages (AM) and by autologous peripheral blood monocytes (PBM). Overall, stimulated production of TNF alpha by AM from lung recipients in vitro was less than that of cells from normal subjects in response to lipopolysaccharide (LPS) challenge, and stimulated production of TNF alpha by AM harvested during conditions of infection or acute and chronic rejection was less than that by cells from healthy lung recipients. AM from normal subjects and allograft recipients produced substantially more TNF alpha than autologous PBM, but release in vitro by PBM from recipients was the same as that from cells of normal subjects who were not immunosuppressed. Thus, systemic immunosuppression does not seem to affect the production of TNF alpha by PBM in vitro, but it may reduce production by AM, indicating different effects of immunosuppression on different compartments of mononuclear cells. This mediator was not detected at elevated levels in serum, and it was undetectable in BAL fluid. We conclude that AM from lung recipients are capable of producing TNF alpha, which would influence the defense and immunogenicity of the allograft.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-4 as a potent down-regulator for human alveolar macrophages capable of producing tumour necrosis factor-alpha and interleukin-1

The effects of recombinant human interleukin-4 (IL-4) on the production of interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF alpha) by human alveolar macrophages (AM) and autologous peripheral blood monocytes (PBM) in response to lipopolysaccharide (LPS) were examined. AM and PBM were obtained by bronchoalveolar lavage and centrifugal elutriation, respectively, from healthy donors. The production of IL-1 (alpha and beta) and TNF alpha by human AM and PBM were quantitated by enzyme immunoassays (EIA). When activated with LPS, AM secreted much more TNF alpha, but less IL-1 beta than PBM. The production of IL-1 (alpha and beta) by activated AM and autologous PBM was suppressed dose-dependently by IL-4. The inhibitory effect of IL-4 was greatest when it was added to AM or PBM simultaneously with LPS or within 3 h after LPS. The suppressive effect of IL-4 was completely neutralized by pretreatment with rabbit anti-IL-4 antiserum. IL-4 also suppressed the production of IL-1 and TNF alpha by monocyte-derived macrophages. As measured by thymocyte co-stimulation assay, the production of cell-associated IL-1 was inhibited by coculture of AM plus LPS with IL-4. Northern blot analysis showed suppression by IL-4 of expression of messenger ribonucleic acid (mRNA) for IL-1 and TNF alpha in LPS-stimulated AM. We conclude that IL-4 is a potent down-regulator for human alveolar macrophages capable of producing IL-1 and TNF alpha.

Different patterns of arachidonate metabolism in autologous human blood monocytes and alveolar macrophages.

As peripheral blood monocytes (PBM) differentiate into tissue macrophages, they undergo a variety of functional changes. One such difference which has been described is an enhanced metabolism of arachidonic acid (AA) via the 5-lipoxygenase (5-LO) pathway in alveolar macrophages (AM) as compared to PBM. In order to elucidate a possible mechanism for this difference, we compared the metabolism of endogenously released AA mobilized by agonists and of exogenously supplied fatty acid in adherent autologous PBM and AM obtained from six normal subjects. Exogenous AA was metabolized to larger amounts of both cyclooxygenase (CO) and 5-LO products by PBM as compared with AM. Although the two cell types released similar amounts of endogenous AA in response to ionophore A23187, marked differences in the pattern of its metabolism were observed. In PBM, a large proportion of released AA remained unmetabolized, and that which was metabolized was converted predominantly to CO products. In contrast, arachidonate released by AM was efficiently metabolized, predominantly via the 5-LO pathway. Similar results were obtained when cells were stimulated with the particulate zymosan, with PBM synthesizing mainly CO and AM, mainly 5-LO eicosanoids. In addition, culture of PBM for up to 5 days in an aerobic environment did not alter their response to A23187 stimulation. These results suggest that the lesser 5-LO metabolism by PBM than AM is not explained by lesser phospholipase or 5-LO activities, but rather a compartmentalization of the endogenous AA deacylated by phospholipase and the 5-LO enzyme in the PBM. The acquisition of the capacity to metabolize endogenous AA to large quantities of 5-LO products as mononuclear phagocytes differentiate in the lung may equip them with the ability to mount an inflammatory response in the alveolar space.

Monocytes stimulate fibroblastoid bone marrow stromal cells to produce multilineage hematopoietic growth factors

In previous studies we have found that monocytes produce soluble factors that stimulate human umbilical vein endothelial cells to produce granulocyte-macrophage colony-stimulating activity (CSA), burst- promoting activity (BPA), and megakaryocyte colony-stimulating activity (Meg-CSA) as well as factors that stimulate T lymphocytes and neonatal fibroblasts to produce CSA. To test the hypothesis that monocytes would similarly stimulate the production of hematopoietic growth factors by autologous bone marrow stromal cells, multiply-passaged adherent fibroblastoid cells derived from the bone marrow of normal volunteers were exposed to conditioned media prepared by incubating autologous peripheral blood monocytes in complete medium for three days. When conditioned media from stromal cells incubated in monocyte-conditioned medium were compared with those of stromal cells cultured in the absence of monocyte-conditioned medium, BPA was increased fourfold and CSA was increased more than 30-fold. We conclude that mononuclear phagocytes recruit stromal cells of the marrow to produce multilineage growth factors in vitro. We suggest that these monocyte-derived recruiting activities may play an important role in orchestration of hematopoietic growth factor production by cells of the marrow microenvironment.

Monocytes Stimulate Fibroblastoid Bone Marrow Stromal Cells to Produce Multilineage Hematopoietic Growth Factors

In previous studies we have found that monocytes produce soluble factors that stimulate human umbilical vein endothelial cells to produce granulocyte-macrophage colony-stimulating activity (CSA), burst-promoting activity (BPA), and megakaryocyte colony-stimulating activity (Meg-CSA) as well as factors that stimulate T lymphocytes and neonatal fibroblasts to produce CSA. To test the hypothesis that monocytes would similarly stimulate the production of hematopoietic growth factors by autologous bone marrow stromal cells, multiply-passaged adherent fibroblastoid cells derived from the bone marrow of normal volunteers were exposed to conditioned media prepared by incubating autologous peripheral blood monocytes in complete medium for three days. When conditioned media from stromal cells incubated in monocyte-conditioned medium were compared with those of stromal cells cultured in the absence of monocyte-conditioned medium, BPA was increased fourfold and CSA was increased more than 30-fold. We conclude that mononuclear phagocytes recruit stromal cells of the marrow to produce multilineage growth factors in vitro. We suggest that these monocyte-derived recruiting activities may play an important role in orchestration of hematopoietic growth factor production by cells of the marrow microenvironment.