Xuan Duc Nguyen

Flow cytometric analysis of T cell proliferation in a mixed lymphocyte reaction with dendritic cells.

BACKGROUND Dendritic cells (DCs) are the most potent antigen-presenting cells. They can be generated in vitro from CD14+ cells, and also from CD34+ progenitor cells. Although T cell proliferation using [3H] thymidine incorporation assay has been used widely to check DC function, this technique only provides limited information about the T cell proliferation. Here, we describe a novel method for quantitative analysis of T cell proliferation using flow cytometry. MATERIALS AND METHODS DCs were generated from CD14+ cells from six healthy blood donors. Monocytes were isolated using positive selection with magnetic cell sorting (MACS) and then cultured with IL-4, GM-CSF, IL-1beta, IL-6, TNF-alpha and PGE(2) to yield fully mature DCs. Allogeneic naive T lymphocytes with known mismatches in HLA classes I and II were cocultured with DCs. Naive T cells without DC stimulation served as negative controls. T cells were harvested on days 0, 3, 5, 7, 9, 11 and analysed by flow cytometry. CD3-ECD and CD4-fluorescein isothiocyanate (FITC) or CD8-FITC antibodies were used to distinguish T cell subsets, whereas T cell activation was measured by assessment of HLA-DR, CD45RO, CD25 and CD71 expression. For T cell quantification, fluorescent microparticles were used. Dead cells were excluded with 7-AAD. The bromdeoxyridine (BrdU)-incorporation ELISA procedure was also performed in order to compare with the T cell proliferation assay with regard to absolute cell counts and CD71 expression. RESULTS The initial T cell concentration on day 1 was 203.9+/-39.7 (173-265) CD3+/CD4+ cells/micro l and 184.5+/-41.6 (148-260) CD3+/CD8+ cells/micro l. The maximal T cell proliferation was recorded on day 7 with a five- to tenfold T cell expansion which resulted in 1994.9+/-383 (1446-2404) CD3+/CD4+ cells/micro l and 944+/-303.7 (560-1483) CD3+/CD8+ cells/micro l. Furthermore, activation markers of both cell lineages were upregulated and reached maxima on days 7 (CD71) and 9 (CD25, HLA-DR). T cell count/micro l as well as CD71 expression both correlated significantly with BrdU incorporation. CONCLUSION Flow cytometric analysis permits simple, precise and rapid quantification of T cell proliferation in a mixed lymphocyte reaction with DCs. Activation, proliferation and cell viability can be simultaneously determined. CD71 is particularly well suited as an activation marker for the simultaneous measurement of T cell proliferation. Thus, specific T cell subsets involved in antigen-specific proliferation can be evaluated in detail.

Endotoxin-induced effects on platelets and monocytes in an in vivo model of inflammation

AimsChronic inflammation is a major contributing factor to atherosclerosis and various markers of inflammation, fibrinolysis and coagulation are upregulated in patients with established atherosclerotic disease. The aim of this study was to investigate the direct and short-term effects of inflammation on platelet and monocyte activation with an in vivo model of endotoxemia in healthy volunteers.Methods and resultsIn this study, 13 healthy male subjects with a mean age of 29.5±5.4 years received intravenous administration of lipopolysaccharide (LPS; 20 IU/kg IV). The kinetics of CD40-ligand and CD62P expression on platelets, tissue-factor binding on monocytes and platelet-monocyte aggregates were measured by whole blood flow cytometry at baseline and at 1, 2, 4, 6 and 24 hours after LPS administration. Plasma levels of soluble CD40-ligand were measured with an ELISA over the same time course. Platelet-monocyte aggregates, tissue-factor binding on monocytes and surface expression of platelet CD40L significantly increased in experimental endotoxemia in vivo, reaching peak values 1 hour after LPS administation. All values returned to baseline after 24 hours. Surface expression of CD62P on platelets and plasma levels of sCD40L did not change significantly in response to LPS.ConclusionsIn vivo administration of endotoxin leads to an activation of platelets and monocytes with an upregulation of proatherogenic CD40L on platelets. These findings underpin the role of inflammation in early atherogenesis through platelet and monocyte activation in an in vivo model.

1α,25-Dihydroxyvitamin D3 Attenuates Platelet Activation and the Expression of VCAM-1 and MT1-MMP in Human Endothelial Cells

Objective: Inflammatory conditions contribute to increased expression of various activity markers in platelets and endothelial cells, leading to atherosclerotic changes in the vascular wall. The objective of this study was to investigate possible protective effects of 1α,25-dihydroxyvitamin D3 in an endothelial cell model. Methods: After a 24-hour incubation with 1α,25-dihydroxyvitamin D3, human umbilical vein endothelial cells were stimulated with lipopolysaccharide (LPS) and incubated in direct contact with platelets. The expression of CD40L and CD62P in platelets, the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 (VCAM-1), the urokinase receptor uPAR and membrane type 1 matrix metalloproteinase (MT1-MMP) in endothelial cells and endothelial cell reactive oxygen species generation were measured by flow cytometry. Endothelial nitric oxide synthase was analyzed by Western blot. Results: The increased expression of VCAM-1 and MT1-MMP in endothelial cells by proinflammatory stimulation with LPS and by direct contact with activated platelets was significantly reduced through preincubation with 1α,25-dihydroxyvitamin D3. Platelets in direct contact with preincubated endothelial cells showed significantly reduced CD62P expression when compared to platelets incubated with untreated endothelial cells. Conclusions: 1α,25-Dihydroxyvitamin D3 attenuates platelet activation and the expression of VCAM-1 and MT1-MMP in human endothelial cells and could have early therapeutic relevance in atherosclerotic diseases.

A novel method for simultaneous analysis of specific platelet antibodies: SASPA

Glycoprotein (GP)‐specific platelet antibodies can cause allo‐immune and auto‐immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet‐specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet‐GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin‐ and fluorescein isotiocyanate‐conjugated goat anti‐human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti‐GP, each with subclasses IgG and IgM) were simultaneously analysed without cross‐reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet‐binding and/or platelet‐associated antibodies were investigated. The monoclonal antibody‐specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet‐specific antibodies (SASPA) assay was able to detect all platelet‐specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet‐specific antibodies.

Simvastatin and atorvastatin attenuate VCAM-1 and uPAR expression on human endothelial cells and platelet surface expression of CD40 ligand.

BACKGROUND In addition to their cholesterol lowering ability, statins have proven pleiotropic effects in the cardiovascular system. Chronic inflammation with interactions between platelets and endothelial cells leads to an upregulation of activity markers of atherosclerosis. The purpose of this study was to investigate the effects of simvastatin and atorvastatin on platelets and endothelial cells in an in vitro endothelial cell model. METHODS AND RESULTS After a 24 h incubation period with either simvastatin or atorvastatin (1 μmol/L), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide (LPS), and were then incubated in direct contact with activated platelets. Platelet surface expression of CD40L and CD62P and expression of ICAM-1, VCAM-1, uPAR and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analyzed by ELISA for soluble MMP-1. The increased expression of VCAM-1 and uPAR on endothelial cells by stimulation with LPS and by direct contact with activated platelets was significantly reduced to a similar extent through pre-incubation with both atorvastatin and simvastatin (p < 0.05). Platelets without endothelial cell contact, but in direct contact with either statin, showed similar significant reductions in surface expression of CD40L (p < 0.005). CONCLUSIONS These effects may explain the ability of statins to reduce the progression of atherosclerosis in addition to their cholesterol-lowering properties.

Screening of human tumor antigens for CD4 T cell epitopes by combination of HLA-transgenic mice, recombinant adenovirus and antigen peptide libraries.

Background As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients. Methods and Findings To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-260–74 epitope and against the new epitope TRP-2149–163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2149–163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1284–298 as a new HLA-DRB1*0301-restricted CD4+ T cell epitope. Conclusions Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives.

IMMUNOHEMATOLOGY: Rapid screening of granulocyte antibodies with a novel assay: flow cytometric granulocyte immunofluorescence test

BACKGROUND: White blood cell (WBC)‐associated antibodies can lead to severe pulmonary transfusion reactions (transfusion‐related acute lung injury [TRALI]). Investigation of a large number of blood donor samples using the standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) proved to be difficult to perform due to the time‐consuming process and the large quantity of test cells required. This study describes the novel flow cytometric GIFT (Flow‐GIFT) method for a rapid detection of granulocyte antibodies by flow cytometric analysis.

Rapid screening of granulocyte antibodies with a novel assay: flow cytometric granulocyte immunofluorescence test.

BACKGROUND: White blood cell (WBC)‐associated antibodies can lead to severe pulmonary transfusion reactions (transfusion‐related acute lung injury [TRALI]). Investigation of a large number of blood donor samples using the standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) proved to be difficult to perform due to the time‐consuming process and the large quantity of test cells required. This study describes the novel flow cytometric GIFT (Flow‐GIFT) method for a rapid detection of granulocyte antibodies by flow cytometric analysis.

The detection of platelet antibodies by simultaneous analysis of specific platelet antibodies and the monoclonal antibody–specific immobilization of platelet antigens: an interlaboratory comparison

BACKGROUND: Glycoprotein (GP)‐specific platelet (PLT) antibodies can cause allo‐ or autoimmune thrombocytopenia. Their detection is of high diagnostic value. The simultaneous analysis of specific PLT antibodies (SASPA) assay is based on simultaneous detection of various PLT‐specific antibodies by flow cytometry and has entered routine use in our Mannheim institution. In this study, we performed an interlaboratory comparison investigation of PLT‐specific antibodies using SASPA versus the “gold standard,” the monoclonal antibody–specific immobilization of PLT antigen (MAIPA) assay.

Differentiation of monocyte-derived dendritic cells under the influence of platelets

BACKGROUND Monocytapheresis has been established to collect a sufficient number of monocytes (MO) for differentiation to dendritic cells (DC) as a cancer vaccine. Platelets (Plt) are invariably found as a contaminant in the final monocytapheresis product. The aim of this study was to investigate DC differentiation under the influence of Plt with regard to their function and phenotype. METHODS MO were isolated and co-cultured with autologous Plt at different MO:Plt ratios (1:1.7, 1:5, 1:15, 1:45 and 1:135) in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-12p70 release after ligation of CD40L was determined in the supernatant by enzyme-linked immunosorbent assay (ELISA). For T-cell stimulation, tetanus toxoid was added to immature DC and maturation was induced by adding cytokines (IL-1beta, IL-6, tumor necrosis factor-alpha and prostaglandin E(2)). Stimulated T cells were analyzed for activation and proliferation as well as for intracellular cytokines by flow cytometry. RESULTS All DC cultures were strongly positive for CD83. At a contaminating concentration of 5 Plt/MO, matured DC showed the highest expression of HLA-DR, CD80 and CD86, inducing a strong T-cell proliferation with high production of IL-4 and interferon-gamma. The highest level of IL-12p70 production was observed by the same DC group. DISCUSSION Plt did not negatively influence DC maturation but enhanced the expression of co-stimulatory molecules and the release of IL-12. Functionally this was reflected by a strong T-cell response that involved T-helper 1 (Th1)- as well as Th2-biased T cells. Our findings show that controlling the Plt concentration may provide important advantages for the generation of DC for use in immunotherapy.