Journal: PLoS ONE

Article Title: Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

doi: 10.1371/journal.pone.0006811

Figure Lengend Snippet: (A) Cell surface loading. HLA-DR4 expressing dendritic cells (DC) generated from the bone marrow of HLA-DR4 transgenic mice were incubated for 4 h with medium alone (left panel) or with 5 µg/ml biotinylated HA 306–318 peptide in the absence (middle panel) or presence of 250 µM AdEtOH, the model MLE compound used throughout this study (right panel). Contour plots are shown for DC after staining with anti-HLA-DR antibody (→ MHC expression) and streptavidin (→ peptide load). Mean peptide loading (MFI of streptavidin signal) is indicated. (B) CD4+ T cell response. DC from HLA-DR4tg mice (left panel) and from HLA-DR1tg mice (right panel) were pulsed for 4 h with indicated amounts of HA 306–318 peptide in the absence (open circle) and presence (closed circle) of 250 µM AdEtOH. The cells were used to challenge HA 306–318 specific, HLA-DR4-restricted 8475/94 cells and HLA-DR1-restricted EvHA/X5 T cell hybridoma cells, respectively. Background proliferation was measured in absence of peptide (dashed line).

Article Snippet: For flow cytometry staining the following were used: α-CD4-PerCP-Cy5.5, α-CD11c-APC (both from BD PharMingen) α-CD154-PE, α-IFNγ-APC (both from Miltenyi), α-HLA-DR-PE (BD Bioscience), α-CD86-Biotin (produced in our laboratory, clone GL1), streptavidin-APC (Caltag).

Techniques: Expressing, Generated, Transgenic Assay, Incubation, Staining

Journal: PLoS ONE

Article Title: Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

doi: 10.1371/journal.pone.0006811

Figure Lengend Snippet: (A) Cell surface loading of MLE-susceptible and –non-susceptible MHC molecules. DC generated from non-susceptible BALB/c mice (left panel) and AdEtOH-susceptible HLA-DR1tg (right panel) were incubated for 4 h with the indicated amounts of biotinylated HA 306–318 peptide in the absence (open circles) or presence (closed circles) of 250 µM AdEtOH. Peptide loading was determined on CD11c+ cells by analyzing the mean fluorescence of the streptavidin-signal gated on a distinct expression of HLA-DR. Background fluorescence was detected in the absence of biotinylated peptide (dashed line). (B) Peptide priming of mice. BALB/c (upper panel) and DR1tg (lower panel) mice were s.c. primed with 100 µg and 3 µg HA 306–318, respectively in IFA/CpG supplemented without (left panel) or with AdEtOH (middle panel). Specific T cell response was determined by intracellular flow cytometry staining at day 12 after priming. Lymph node cells were incubated for 6 hrs in the presence of 10 µg HA 306–318 or 10 µg MOG 35–55 as irrelevant control peptide (right panel) and αCD28 antibody. 3 µg/ml Brefeldin A was added for the last 2 h. Intracellular IFNγ-production was analyzed on CD4+ CD154+ double positive T cells. Numbers indicate frequency of CD4+ CD154+ IFNγ+ cells among total CD4+ cells. Data representative of at least two independent experiments are shown.

Article Snippet: For flow cytometry staining the following were used: α-CD4-PerCP-Cy5.5, α-CD11c-APC (both from BD PharMingen) α-CD154-PE, α-IFNγ-APC (both from Miltenyi), α-HLA-DR-PE (BD Bioscience), α-CD86-Biotin (produced in our laboratory, clone GL1), streptavidin-APC (Caltag).

Techniques: Generated, Incubation, Fluorescence, Expressing, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

doi: 10.1371/journal.pone.0006811

Figure Lengend Snippet: (A) Cell surface loading of NY-ESO-1 epitopes. L929 fibroblasts transfected with HLA-DR1 (left panel) or HLA-DR4 (right panel) were incubated for 4 h with titrated amounts of NY-ESO-1 89–101 or NY-ESO-1 119–143, respectively. Loading was performed in the absence (closed circles) or presence (open circles) of 250 µM AdEtOH. Non-transfected L929 cells were used as a negative control (left side). Peptide loading was determined by analyzing the mean fluorescence of the streptavidin-signal gated on a distinct expression of HLA-DR. Background fluorescence was detected in the absence of biotinylated peptide (dashed line). (B) Detection of tumour-specific T cell response in vivo . Groups of HLA-DR1tg (left panel, n = 13) or HLA-DR4tg (right panel, n = 10) mice were s.c. primed with 5 µg of the respective NY-ESO-1 epitopes in IFA/CpG supplemented with or without AdEtOH. 12 days after vaccination, 1×10 6 Lymph node cells were incubated with titrated amounts of NY-ESO-1 89–101 or NY-ESO-1 119–143, respectively. IFNγ-detection was carried out 48 hrs later using an Elispot assay and summarized data were analyzed using student's t test.

Article Snippet: For flow cytometry staining the following were used: α-CD4-PerCP-Cy5.5, α-CD11c-APC (both from BD PharMingen) α-CD154-PE, α-IFNγ-APC (both from Miltenyi), α-HLA-DR-PE (BD Bioscience), α-CD86-Biotin (produced in our laboratory, clone GL1), streptavidin-APC (Caltag).

Techniques: Transfection, Incubation, Negative Control, Fluorescence, Expressing, In Vivo, Enzyme-linked Immunospot

Journal: Stem Cell Research & Therapy

Article Title: Osteogenic differentiation of mesenchymal stem cells promotes c-Jun-dependent secretion of interleukin 8 and mediates the migration and differentiation of CD4 + T cells

doi: 10.1186/s13287-022-02735-0

Figure Lengend Snippet: Phenotype identification and trilineage differentiation potential of bone marrow-derived mesenchymal stem cells (MSCs). a MSCs were negative for CD14, CD45 and HLA-DR expression and positive for CD29, CD44 and CD105 expression. b MSCs were successfully induced to undergo osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. Scale bars in the osteogenesis and adipogenesis images, 250 μm. Scale bar in the chondrogenesis image, 100 μm

Article Snippet: Collected MSCs were incubated in the dark for 25 min at room temperature with the following surface marker-specific antibodies: anti-human CD14-PE (BD, 562691), CD34-PE (BD, 551387), HLA-DR-PE (BD, 555812), CD73-FITC (BD, 561254), CD90-FITC (BD, 555595) and CD105-FITC (BD, 561443).

Techniques: Derivative Assay, Expressing

Journal: Oncoimmunology

Article Title: Exploiting a new strategy to induce immunogenic cell death to improve dendritic cell-based vaccines for lymphoma immunotherapy

doi: 10.1080/2162402X.2017.1356964

Figure Lengend Snippet: RA/IFNα-TCL pulsed-IFN-DCs induce tumor-specific CTLs with enhanced killing efficiency. (A) HLA-A*0201-restricted healthy donor-derived lymphoma-specific CTL cultures were able to efficiently lyse MCL cell lines. The incubation of target cells with the anti-HLA A*0201 cr11.351 mAb prevented almost completely the lysis mediated by CTLs primed with RA/IFNα-TCL pulsed-DCs. Only background lysis was observed for CTLs cultured with unpulsed-DCs, CNTR-TCL or γ-radiated-TCL pulsed-DCs in all performed tests. (p < 0.05, Student's t-test). (B) HLA-B*0801-restricted tumor-specific CTLs from different healthy donors were generated with IFN-DCs loaded either with CNTR-TCLs, RA/IFNα-TCLs and γ-rad-TCLs obtained from DOHH2 DLBCL cell line. (p < 0.02, Student's t-test). (C) HLA-A*0201 healthy donor-derived tumor specific CTLs were able to specifically recognize and lyse autologous cells presenting 5 HLA-A*0201-restricted cyclin D1-derived peptides. Peptide-loaded T2-A2 cells were used as target cells and CTL cultures were obtained co-culturing (left) Mino cell line lysate-loaded and (right) SP53 cell line lysate-loaded IFN-DCs with autologous T lymphocytes. D) HLA-B*0801 healthy donor-derived tumor-specific CTLs were able to specifically recognize and kill autologous EBV-LCLs presenting several HLA-B*0801-restricted Survivin-derived peptides. Peptide-loaded autologous EBV-LCLs were used as target cells and CTLs were generated by co-culturing DOHH2 TCL-loaded IFN-DCs with donor's T lymphocytes. (p < 0.05, Student's T-test). All cytotoxicity assays, evaluated by standard calcein-AM release assay, were performed after 4 re-stimulations by using as target cells either TCL-corresponding tumor cell lines and peptide-loaded T2-A2 cells/autologous LCLs. The graphs represent a mean of 3 independent experiments from different healthy donors. All tests were performed in triplicate at E:T ratio of 10:1, when tumor cell lines were used as targets, and at E:T of 20:1 in peptide-specific cytotoxicity assays. (* vs unpulsed-DCs; ** vs CNTR-TCL pulsed-DCs).

Article Snippet: CD3 FITC (mouse immunoglobulin (Ig) G1, clone UCHT1), CD4 PeCy7 (Mouse BALB/c IgG 1 , SK3) and APC-Cy7 (mouse IgG1, RPA-T4), CD8 BV421 (mouse IgG1, RPA-T8), CD25 APC (mouse IgG1, M-A251), CD45RA AlexaFluor700 (mouse IgG2b, HI100), CD127 PerCP-Cy5.5 (mouse IgG1, HIL-7R-M21), Ki-67 Pe-Cy7 (mouse IgG1, B56), TNF-alpha PE and PerCP-Cy5.5 (mouse IgG1, MAb11), IFN-gamma PeCy7 (mouse IgG1, 4 S.B3), IL-17 AlexaFluor647 (mouse IgG1, N49-653), HLA DR-PE (555812), and CD47-AlexaFluor647 (561249) were purchased from BD Biosciences (Becton Dickinson), CD1 a-PE (mouse IgG1, BL6), CD3-ECD (mouse IgG1, UCHT1), CD11 c-PE (IM1760), CD11 c-PC5 (IM3707), CD25-ECD (mouse IgG2 a, B1.49.9), CD80-PE (IM1976U), CD83-PE (IM2218), CD86-PE (182729) are from Beckman Coulter, CD127-Pe-Cy5 (mouse IgG1, eBioRDR5), FoxP3 PE (mouse IgG1, 236/E7), IL-17 FITC (mouse IgG1, eBio64DEC17), and IL-22 PerCP-eFluor710 (mouse IgG1, 22URTI) were from eBioscience (Affymetrix), HSP70 (ADI-SPA-810PE) and HSP90 (ADI-SPA-830-488) were from Enzo Life Science, anti-rabbit-PE (111-116-144) was from Jackson ImmunoReseach and anti-mouse-FITC secondary antibody is from Beckman Coulter (IM1619).

Techniques: Derivative Assay, Incubation, Lysis, Cell Culture, Generated, Release Assay

Journal: Oncoimmunology

Article Title: Exploiting a new strategy to induce immunogenic cell death to improve dendritic cell-based vaccines for lymphoma immunotherapy

doi: 10.1080/2162402X.2017.1356964

Figure Lengend Snippet: Generation of specific human CD3+ T cells in hu-PBL-NOD-scid mice immunized with TCL pulsed-DCs and evaluation of tumor-growth inhibition. (A) Immunization schedule for hu-PBL-NOD-scid mice vaccination. Mice were injected i.p. with 40 × 106 PBL from HLA-A2 healthy blood donors. Four days after reconstitution, hu-PBL-SCID mice were injected i.p. with autologous IFN-DC loaded with TCL (200 μg). Vaccinated mice received boost immunizations after 7 and 14 days and were sacrificed 7 days later. Evaluation of CD3+ T cell response by ELISPOT assay for IFN-gamma and Granzyme B. Human cells recovered from four spleens of hu-PBL-SCID mice from each group were pooled. The assay was performed using as stimulators MCL cells Mino. Bars represent the CD3+ T cell response from hu-PBL-NOD-scid mice immunized with either RA/IFNα-TCL pulsed-DCs or CNTR-TCL pulsed-DCs as compared to the basal CD3+ T cell response from hu-PBL-NOD/SCID mice injected with unloaded IFN-DCs. Bars represent the mean ± SD of 3 independent experiments. (B) Evaluation of tumor-growth inhibition after therapeutic vaccination schedule. Graphs represent Mino cell growth rate in hu-PBL-NOD-scid mice immunized as described in materials and methods (*p < 0.05 Student's t-test). (C) Evaluation of therapeutic vaccination effect on body weight in tumor-bearing hu-PBL-NOD-scid mice. Data represent the mean body weight in the different treatment group. One representative experiment is shown. (D) Assessment of IFNγ levels in serum of mice after therapeutic vaccination schedule (*p < 0.05 Student's t-test).

Article Snippet: CD3 FITC (mouse immunoglobulin (Ig) G1, clone UCHT1), CD4 PeCy7 (Mouse BALB/c IgG 1 , SK3) and APC-Cy7 (mouse IgG1, RPA-T4), CD8 BV421 (mouse IgG1, RPA-T8), CD25 APC (mouse IgG1, M-A251), CD45RA AlexaFluor700 (mouse IgG2b, HI100), CD127 PerCP-Cy5.5 (mouse IgG1, HIL-7R-M21), Ki-67 Pe-Cy7 (mouse IgG1, B56), TNF-alpha PE and PerCP-Cy5.5 (mouse IgG1, MAb11), IFN-gamma PeCy7 (mouse IgG1, 4 S.B3), IL-17 AlexaFluor647 (mouse IgG1, N49-653), HLA DR-PE (555812), and CD47-AlexaFluor647 (561249) were purchased from BD Biosciences (Becton Dickinson), CD1 a-PE (mouse IgG1, BL6), CD3-ECD (mouse IgG1, UCHT1), CD11 c-PE (IM1760), CD11 c-PC5 (IM3707), CD25-ECD (mouse IgG2 a, B1.49.9), CD80-PE (IM1976U), CD83-PE (IM2218), CD86-PE (182729) are from Beckman Coulter, CD127-Pe-Cy5 (mouse IgG1, eBioRDR5), FoxP3 PE (mouse IgG1, 236/E7), IL-17 FITC (mouse IgG1, eBio64DEC17), and IL-22 PerCP-eFluor710 (mouse IgG1, 22URTI) were from eBioscience (Affymetrix), HSP70 (ADI-SPA-810PE) and HSP90 (ADI-SPA-830-488) were from Enzo Life Science, anti-rabbit-PE (111-116-144) was from Jackson ImmunoReseach and anti-mouse-FITC secondary antibody is from Beckman Coulter (IM1619).

Techniques: Inhibition, Injection, Enzyme-linked Immunospot

Journal:

Article Title: Regulation of STAT pathways and IRF1 during human dendritic cell maturation by TNF-? and PGE2

doi: 10.1189/jlb.0107040

Figure Lengend Snippet: Cell surface phenotype of human monocyte-derived, immature DCs (iDC) and mature DCs (mDC). DCs were generated as described in Materials and Methods, and the expression of CD14, CD40, CD25, CD80, CD86, and HLA-DR was analyzed by flow cytometry after 2 days of stimulation with 10 ng/ml LPS or TNF-α (25 ng/ml) + PGE2 (1 ng/ml). Open histograms = isotype control staining; shaded histograms = staining with corresponding antibodies. One representative experiment of seven is shown.

Article Snippet: Reagents and antibodies CD14-FITC, CD25-PE, CD40-FITC, CD80-PE, CD86-PE, and their corresponding isotype control antibodies were from BD PharMingen (San Diego, CA, USA), and HLA-DR-PE was from Immunotech (Beckman Coulter, Fullerton, CA, USA).

Techniques: Derivative Assay, Generated, Expressing, Flow Cytometry, Control, Staining

Journal:

Article Title: Regulation of STAT pathways and IRF1 during human dendritic cell maturation by TNF-? and PGE2

doi: 10.1189/jlb.0107040

Figure Lengend Snippet: TNF-α + PGE2-induced IRF1 expression is partially dependent on NF-κB but not on IFN-α/β. (A) mRNA levels of IRF1 in immature DC treated with LPS, TNF-α, PGE2, or TNF-α + PGE2 for indicated times were measured by real-time PCR and normalized relative to GAPDH. (B) mRNA levels of IRF1 in immature DC treated with TNF-α, PGE2, or TNF-α + PGE2, with or without blocking anti-IFNR-α/β antibodies, were measured by real-time PCR and normalized relative to GAPDH. (C) mRNA levels of IRF1 in immature DC treated with TNF-α, PGE2, or TNF-α + PGE2, with or without the IKK inhibitor Bay11, were measured by real-time PCR and normalized relative to GAPDH. (D) The expression of CD25, CD40, CD86, and HLA-DR was analyzed by flow cytometry after 48 h of stimulation with TNF-α (25 ng/ml) + PGE2 (1 ng/ml), with or without Bay11 treatment. Open histograms with dotted line = isotype control staining; shaded histograms = staining with corresponding antibodies without Bay11 treatment; and open histograms with solid line = staining with corresponding antibodies in immature DC treated with Bay11. One representative experiment of two is shown.

Article Snippet: Reagents and antibodies CD14-FITC, CD25-PE, CD40-FITC, CD80-PE, CD86-PE, and their corresponding isotype control antibodies were from BD PharMingen (San Diego, CA, USA), and HLA-DR-PE was from Immunotech (Beckman Coulter, Fullerton, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Blocking Assay, Flow Cytometry, Control, Staining

Journal:

Article Title: Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions

doi:

Figure Lengend Snippet: In situ expression of CD1 molecules and HLA-DR in primary and metastatic melanoma. Immunohistochemistry was performed to study in vivo CD1 expression in primary (A and B) and metastatic (C and D) melanoma lesions using the APAAP technique. Sections were counterstained with hematoxylin. Both A and C show CD1c expression. HLA-DR expression in primary and metastatic lesions is shown in B and D. Pictures shown are representative stainings. Original magnification, ×200. E: Confocal microscopy shows co-expression of CD1 molecules and HLA-DR in primary but not metastatic melanoma. To demonstrate co-expression of CD1 molecules and HLA-DR on melanoma cells as well as CD1 down-regulation in metastatic melanoma lesions, confocal microscopy was performed. Pictures show CD1b (green), HLA-DR (red), and CD1b/HLA-DR co-expression (yellow) and are representative stainings. Top row, primary melanoma; bottom row, metastatic melanoma. The horizontal bar indicates 20 μm.

Article Snippet: 14,30 The antibodies used were against CD1a (IgG 2a ) (Dako, Zug, Switzerland), CD1b (BCD1b3.1.6, IgG 1 ) kindly provided by Dr. S. A. Porcelli (Harvard Medical School, Boston, MA, USA), CD1c (IgG 1 ) (Immunotech, Marseilles, France), CD1d (IgG 1 ) (Biosource International, Camarillo, CA, USA), CD1d (51.1.3, IgG 2b ), kindly provided by Dr. S. P. Balk (Harvard Medical School), HLA-DR (Becton Dickinson) (all at 10 μg/ml), and IL-10 (5 μg/ml) (R&D Systems).

Techniques: In Situ, Expressing, Immunohistochemistry, In Vivo, Confocal Microscopy

Journal:

Article Title: Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions

doi:

Figure Lengend Snippet: To assess the number of cells expressing CD1a, b, c, d in melanoma lesions, primary (n = 10) and metastatic (n = 10) melanoma serial cryosections were stained using the respective antibodies and the APAAP technique. The number of CD1a, b, c and HLA-DR-positive cells was thereafter counted in five different microscopic fields (original magnification, ×400) and the mean values were calculated. Results are given as the ratio between CD1a, b, c- and HLA-DR-positive cells to correct for the density of infiltrating DC. *, P < 0.05, in relation to the primary melanoma group.

Article Snippet: 14,30 The antibodies used were against CD1a (IgG 2a ) (Dako, Zug, Switzerland), CD1b (BCD1b3.1.6, IgG 1 ) kindly provided by Dr. S. A. Porcelli (Harvard Medical School, Boston, MA, USA), CD1c (IgG 1 ) (Immunotech, Marseilles, France), CD1d (IgG 1 ) (Biosource International, Camarillo, CA, USA), CD1d (51.1.3, IgG 2b ), kindly provided by Dr. S. P. Balk (Harvard Medical School), HLA-DR (Becton Dickinson) (all at 10 μg/ml), and IL-10 (5 μg/ml) (R&D Systems).

Techniques: Expressing, Staining

Journal:

Article Title: Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions

doi:

Figure Lengend Snippet: IL-10 down-regulates CD1 expression on monocyte-derived DC in a dose-dependent manner. Monocyte-derived DC were cultured for 3 days in the presence of IL-4 (1000 IU/ml) and GM-CSF (800 IU/ml). Monocyte-derived DC were thereafter cultured for another 72 hours in GM-CSF and IL-4 in the presence of various IL-10 concentrations (1, 10, and 100 ng/ml). A: Analysis of CD1a, b, c and CD86, CD80, and (B) HLA-DR expression by flow cytometry. Monocyte-derived DC cultured in presence of GM-CSF and IL-4 without IL-10 were used as positive control. Results are representative for three independent experiments. Filled histograms represent isotype controls.

Article Snippet: 14,30 The antibodies used were against CD1a (IgG 2a ) (Dako, Zug, Switzerland), CD1b (BCD1b3.1.6, IgG 1 ) kindly provided by Dr. S. A. Porcelli (Harvard Medical School, Boston, MA, USA), CD1c (IgG 1 ) (Immunotech, Marseilles, France), CD1d (IgG 1 ) (Biosource International, Camarillo, CA, USA), CD1d (51.1.3, IgG 2b ), kindly provided by Dr. S. P. Balk (Harvard Medical School), HLA-DR (Becton Dickinson) (all at 10 μg/ml), and IL-10 (5 μg/ml) (R&D Systems).

Techniques: Expressing, Derivative Assay, Cell Culture, Flow Cytometry, Positive Control

Journal:

Article Title: Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions

doi:

Figure Lengend Snippet: Effects of melanoma supernatants on CD1, CD86, and HLA-DR expression on monocyte-derived DC. A: Supernatants from first passage metastatic melanoma cultures with high (cell line 000630, 306 pg/ml IL-10) and low (cell line 000515, 91 pg/ml IL-10) IL-10 concentrations were used to study the effects of different supernatant dilutions on CD1 expression on GM-CSF/IL-4-generated DC. After 3 days, 0%, 10%, 50%, and 100% of the medium was replaced with melanoma supernatants from the melanoma cell culture indicated and incubated for another 3 days. CD1a, b, and c expression was thereafter assessed by flow cytometry. B: CD86, CD80, and HLA-DR expression on DC after replacement of 0%, 10%, 50%, and 100% of DC culture medium with melanoma supernatant (cell line 000630). C: Supernatants from different melanoma and fibroblast cell cultures were used to study the effects on CD1 expression on DC using flow cytometry. Fifty percent of the DC culture medium was replaced with first passage metastatic melanoma (culture 000630), second passage metastatic melanoma (culture 000621), primary melanoma (culture 990220), and fibroblast cell culture supernatants. CD1 expression was thereafter measured using flow cytometry. Results are shown as the mean MFI of three independent experiments. Vertical bars represent the standard deviations of the means. *, P < 0.05, in relation to monocyte-derived DC cultured in the absence of cell culture supernatant. MM, metastatic melanoma; PM, primary melanoma; fibro, fibroblasts; P, passage.

Article Snippet: 14,30 The antibodies used were against CD1a (IgG 2a ) (Dako, Zug, Switzerland), CD1b (BCD1b3.1.6, IgG 1 ) kindly provided by Dr. S. A. Porcelli (Harvard Medical School, Boston, MA, USA), CD1c (IgG 1 ) (Immunotech, Marseilles, France), CD1d (IgG 1 ) (Biosource International, Camarillo, CA, USA), CD1d (51.1.3, IgG 2b ), kindly provided by Dr. S. P. Balk (Harvard Medical School), HLA-DR (Becton Dickinson) (all at 10 μg/ml), and IL-10 (5 μg/ml) (R&D Systems).

Techniques: Expressing, Derivative Assay, Generated, Cell Culture, Incubation, Flow Cytometry