Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: Work panel used and monoclonal antibody/dye information.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques:

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: AEA patient's non-activated CD4 + T cells show increased surface expression of CD46, which is dependent on pollen season. Isolated CD4 + T cells from 49 HDs and 58 patients with AEA were analyzed non-activated (NA). The surface expression of CD46 on NA CD4 + T cells was assessed in HDs, AEA in LPP and HPP (A) as well as in HDs vs. AEA based on disease severity in LPP (B) and HPP (C) . Data were measured by flow cytometry and are presented as median fluorescence intensity (MFI) in graphs (A–C) . Horizontal bars represent the median. Next, the surface expression of CD46 was analyzed in HDs and AEA CD4 + T cells under both non-activated (NA) conditions (D) and after stimulation with αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and a high dose of IL-2 (50 U/ml), depending on LPP and HPP (E) . Concentration of sCD46 was assessed in plasma samples using ELISA in 40 HDs and 40 AEA patients in both LPP and HPP (F) . Data are presented as a median +95% CI in graphs (D) , (E) and (F) . Statistical analysis was performed using the unpaired Mann–Whitney U -test for two-group comparisons, and the Kruskal–Wallis test with Dunn's correction for three or more comparisons within a single graph where appropriate; ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. HDs, healthy donors; AEA, allergic eosinophilic asthma; NA, non-activated; LPP, low pollen period; HPP, high pollen period; MFI, median fluorescence intensity; CI, confidence interval.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Isolation, Flow Cytometry, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: AEA patient's CD4 + T cells show increased expression of CD46 after stimulation in low pollen period. CD4 + T cells from 49 HDs and 58 patients with AEA were cultured with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46/IL-2) for 60 h. (A) Surface expression of CD46 on CD4 + T cells was compared in both HDs and AEA patients in dependence of LPP and HPP, as well as between AEA patients in dependence of LPP and HPP (B) . Serum ECP levels were correlated with percentage of CD46 + CD4 + T cells after αCD3/αCD46/IL-2 stimulation among HDs, AEA LPP and AEA HPP (C) . Surface expression of CD46 was measured by flow cytometry and is presented as median fluorescence intensity (MFI). Statistical analysis of CD46 expression was performed using Kruskal–Wallis test with Dunn's correction for multiple comparisons all vs. all. Correlation analysis was performed using non-parametric Spearman correlation coefficient; ns (not significant), * p ≤ 0.05, ** p ≤ 0.01. HDs, healthy donors; AEA, allergic eosinophilic asthma; mAbs, monoclonal antibodies; LPP, low pollen period; HPP, high pollen period; ECP, eosinophilic cationic protein; MFI, median fluorescence intensity.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: CD4 + T cells differ in CD46 downregulation after calcitriol co-stimulation in both HDs and AEA patients. CD4 + T cells from 49 HDs and 58 patients with AEA were cultured without stimuli (NA) or with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46) for 60 h alone or with calcitriol (1 × 10 −7 M). The surface expression of CD46 was analyzed before/after calcitriol stimulation in HDs (A) and AEA patients in LPP (B) . Subsequently, two different groups were identified based on CD46 expression after calcitriol stimulation based on following formula: x = MFI CD46 (αCD3/αCD46/IL-2/Calcitriol)—MFI CD46 (αCD3/αCD46/IL-2). When x < 0 = Group CD46D (Decrease) (A1,B1) , when x > 0 = Group CD46I (Increase) (A2,B2) . Representative histograms depicts surface CD46 expression on NA or activated CD4 + T cells in groups CD46D/CD46I in HDs (C) and AEA patients (D) . Statistical analysis was performed using Kruskal–Wallis test with Dunn's correction for multiple comparisons all vs. all; ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. HDs, healthy donors; AEA, allergic eosinophilic asthma; NA, non-activated; mAbs, monoclonal antibodies; LPP, low pollen period; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Cell Culture, Expressing

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: CD46D and CD46I groups of HDs and AEA patients differ in CD46 expression on both non-activated and stimulated CD4 + T cells. CD4 + T cells from 49 HDs and 58 patients with AEA were cultured without stimuli (NA) or with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46/IL-2) and with calcitriol (1 × 10 −7 M) (αCD3/αCD46/IL-2/Cal). Surface expression of CD46 was measured in both groups CD46D/CD46I from HDs (A) , as well as from AEA patients (B) . Subsequently, percentage of CD46 downregulation after αCD3/αCD46/IL-2/Calcitriol stimulation was assessed in the group CD46D from HDs and AEA patients (C) . Identically, percentage of CD46 upregulation after αCD3/αCD46/IL-2/Calcitriol stimulation was assessed in the CD46I group from HDs and AEA patients (D) . The surface expression of CD46 was measured by flow cytometry and is presented as median fluorescence intensity (MFI) in graphs (A) and (B) . Graphs (C) and (D) are depicted as median + 95% CI. Statistical analysis was performed using the non-parametric Mann–Whitney U test; ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. HDs (healthy donors), AEA, allergic eosinophilic asthma; NA, non-activated; mAbs, monoclonal antibodies; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation; MFI, median fluorescence intensity; CI, confidence interval.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: Increased CD46 expression on CD4 + T cells from both HDs and AEA patients is independent of serum 25-OH vitamin D concentration. To clarify the relationship between the surface expression of CD46 on NA CD4 + T cells and serum levels of 25-OH vitamin D, these parameters were correlated in both HDs ( n = 49) (A) and AEA patients in LPP ( n = 58) (B) , as well as in HPP (C) . Next, serum levels of 25-OH vitamin D were compared between HDs and AEA (D) as well as in dependence on asthma severity (E) . The serum concentration of 25-OH vitamin D was also compared after dividing HDs and AEA patients into CD46D/CD46I groups (F) . Correlation analysis was performed using the non-parametric Spearman correlation coefficient, comparison of serum 25-OH vitamin D levels between HDs and AEA was performed using the non-parametric Mann–Whitney U -test, while the Kruskal–Wallis test with Dunn's correction was used for multiple comparisons all vs. all; ns (not significant), * p ≤ 0.05. HDs, healthy donors; AEA, allergic eosinophilic asthma; NA, non-activated; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol co-stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol co-stimulation.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Concentration Assay, Comparison, MANN-WHITNEY

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: AEA patients in low pollen period show increased CD25 expression on stimulated CD4 + T cells, which is further promoted by calcitriol. CD4 + T cells from 49 HDs and 58 patients with AEA were cultured with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46/IL-2) or with calcitriol (1 × 10 −7 M) (αCD3/αCD46/IL-2/Cal) for 60 h. (A) Surface expression of CD25 (IL-2 receptor α chain) and proliferation (B) were analyzed by flow cytometry before/after calcitriol stimulation with αCD3/αCD46/IL-2 in HDs and AEA patients in LPP and HPP groups using the non-parametric Mann–Whitney U -test. The data are presented as median intensity fluorescence (MFI), where horizontal bars indicate the median. Based on the CD46 dynamics after calcitriol stimulation, HDs and AEA patients were divided into groups CD46D/CD46I and surface expression of CD25 was analyzed separately. (C) To better understand the effect of calcitriol on CD25 expression in CD4 + T cells between HDs and AEA patients, we expressed the results as the percentage of upregulation after stimulation. Next, sIL-2RA levels were assessed in cell culture SNs, with HDs and AEA LPP patients divided into CD46D/CD46I groups (E) and are presented as a median +95% CI. Data from panels (C) and (E) were analyzed using the Wilcoxon-sign rank test and data from graphs (D) and (F) using the Kruskal–Wallis test with Dunn's correction; ns (not significant), * p ≤ 0.05. HDs, healthy donors; AEA, allergic eosinophilic asthma; mAbs, monoclonal antibodies; LPP, low pollen period; sIL-2RA, soluble IL-2 receptor α chain; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation; MFI, median fluorescence intensity; CI, confidence interval.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Cell Culture, Flow Cytometry, MANN-WHITNEY, Fluorescence

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: Stimulated CD4 + T cells from CD46D group produce significantly more IFN-γ and IL-10 in HDs and react to calcitriol co-stimulation with downregulation of IFN-γ and upregulation of IL-10. CD4 + T cells from 49 HDs were cultured with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46/IL-2) or with calcitriol (1 × 10 −7 M) (αCD3/αCD46/IL-2/Cal) for 60 h. The concentrations of IFN-γ (A) and IL-10 (B) were assessed using ELISA and analyzed in accordance with the division of HDs into CD46D/CD46I groups. The results are presented as the median +95% CI. Similarly, the percentages of IFN-γ + CD4 + T cells (C) and IL-10 + CD4 + T cells (D) obtained from flow cytometry were analyzed, with the horizontal bar representing the median. Data were analyzed using the Kruskal–Wallis test with Dunn's correction for multiple comparisons all vs. all. (E) The timeline showing IFN-γ and IL-10 production by αCD3/αCD46/IL-2-stimulated CD4 + T cells at 0, 12, 36, 60 and 84 h of incubation is based on data from three healthy donors and is presented as the median + 95% confidence interval. (F) Representative dot-plots depict differences in IFN-γ and IL-10 production by activated CD4 + T cells respecting the division into the groups CD46D/CD46I. The data from flow cytometry were obtained from CD4 + T cells treated with Brefeldin A during the final 4 h of stimulation and were gated from 90.000 CD4 + T cells. The numbers in the top right corner show percentage of CD4 + T cells in each quadrant. The data are representatives of 107 individual samples performed in 18 series. HDs, healthy donors; mAbs, monoclonal antibodies; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Incubation, Expressing

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: AEA patient's CD4 + T cells from CD46D group show increased production of IFN-γ after stimulation, which can be downregulated by calcitriol and simultaneously produce more IL-10. CD4 + T cells from 49 HDs and 58 AEA patients were cultured with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/ α CD46/IL-2) or with calcitriol (1 × 10 −7 M) (αCD3/αCD46/IL-2/Cal) for 60 h. Concentrations of IFN-γ and IL-10 were measured in cell culture SNs using ELISA. IFN-γ production was compared between AEA LPP and AEA HPP groups (A) as well as in groups CD46D (B) and CD46I (C) . An identical analysis was performed with production of IL-10 (D–F) . To simplify the evaluation of the calcitriol's effect in both HDs and AEA patients, with respect to the CD46D/CD46I group division, results are presented as the percentage of downregulation in IFN-γ (G) and percentage of upregulation in IL-10 (H) after αCD3/αCD46/IL-2/Cal stimulation. Results are presented as the median + 95% CI. Paired data were analyzed using Wilcoxon matched-pairs signed rank test (black color) and unpaired data were analyzed unpaired Mann–Whitney U -test (grey color); ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. HDs, healthy donors; AEA, allergic eosinophilic asthma; mAbs, monoclonal antibodies; SNs, supernatant; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation; CI, confidence interval.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Expressing

Journal: Virology

Article Title: Receptor usage of a newly emergent adenovirus type 14.

doi: 10.1016/j.virol.2009.02.034

Figure Lengend Snippet: Fig. 2. Competition of viruses for attachment on 293 cells. (A and B) 293 cells were pre-incubated with Ad14-de Wit, Ad14a, Ad3, Ad35 or 10 μg/ml CD46 antibody (MEM-258, Serotec) for 1 h on ice. After washing, 3H-Ad14-de Wit, (A, left panel), 3H-Ad14a, (A, right panel), 3H-Ad3, (B, left panel) or 3H-Ad35 (B, right panel) was added for another hour on ice. After washing, cell-associated radioactivity was determined. N=3. Bars indicate the means. Standard deviation was less than 10% in all cases. (C) Affinity of Ad14-de Wit and Ad14a to 293 cells. To generate Scatchard blots, cells were incubated with increasing MOIs of 3H-Ad14-de Wit or 3H-Ad14a and the number of cells associated viral particles was measured after 1 h of incubation on ice. The y-axis shows the ratio between bound particles to total input particles minus bound particles. The x-axis shows the number of bound particles. The binding affinities (Ka) of virus were calculated on the basis of the slope with standard Excel software as described previously (Tuve et al., 2006).

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Incubation, Radioactivity, Standard Deviation, Binding Assay, Virus, Software

Journal: Virology

Article Title: Receptor usage of a newly emergent adenovirus type 14.

doi: 10.1016/j.virol.2009.02.034

Figure Lengend Snippet: Fig. 3. Fiber knob competition Ad14-de Wit and Ad14a attachment. (A) Analysis of recombinant Ad fiber knobs. Purified Ad35, Ad14-de Wit, and Ad14a knob proteins (1 μg/lane) were run as native protein (N) or after denaturation (D) on a polyacrylamide gel. Coomassie brilliant blue staining (left panel) revealed the trimeric knob form, which is converted into monomers after boiling. Western blots (right panel) were analyzed for CD46 binding to fiber knobs by subsequent incubation with sCD46, anti-CD46 Mab, and anti-mouse IgG- HRP. (B) Fiber knob competition. Cells were pre-incubated with 0.4 μg or 4 μg knob protein on ice for 1 h. Then 3H-Ad14-de Wit or 3H-Ad14a virus was added and cell-associated radioactivity was measured after 1 hour incubation. N=3. Bars indicate the means. Standard deviation was less than 10% in all cases.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Recombinant, Staining, Western Blot, Binding Assay, Incubation, Virus, Radioactivity, Standard Deviation

Journal: Blood Advances

Article Title: In vivo HSC transduction in rhesus macaques with an HDAd5/3 + vector targeting desmoglein 2 and transiently overexpressing cxcr4

doi: 10.1182/bloodadvances.2022007975

Figure Lengend Snippet: Analysis of DSG2 expression on HSCs. (A) Flow cytometry for CD46 and DSG2 on human CD34 + cells from 2 different healthy G-CSF–mobilized donors. (B) Schematic of HDAd vector capsids and genomes. The chimeric capsids contain either affinity-enhanced Ad35 or Ad3 fiber knob domains. Both vector genomes contain an EF1α-mgmt p140k /GFP expression cassette used before. Both proteins are linked via a self-cleaving picornavirus 2A peptide. One set of vectors has an additional CMV promoter–human cxcr4 expression cassette. The 4 vectors used in this study are HDAd5/35++GFP, HDAd5/3+GFP, HDAd5/35++GFP/cxcr4, and HDAd5/3+GFP/cxcr4. (C) In vitro CD34 + cell transduction studies. Cells were infected with HDAd5/35++-GFP and HDAd5/3+-GFP virus at a multiplicity of infection of 2000 vp/cell with and without pre-incubation with recombinant soluble CD46 or DSG2 (10 μg/mL) for 1 hour. GFP expression was analyzed 24 hours' postinfection. (D) CD46 and DSG2 flow cytometry on human and rhesus peripheral RBCs stained with antibodies that recognize the receptors in both species (anti-human CD46 mAb clone M177 from Santa Cruz Biotechnology [Dallas, TX] and anti-human DSG2 polyclonal antibody AF947 from R&D Systems [Minneapolis, MN]). (E) Transduction of 293 cells with HDAd5/35++GFP and HDAd5/3+GFP in the absence and presence of human (huRBCs) and rhesus (rhRBCs) RBCs (details are provided in the Materials and methods). Shown is the percentage of GFP-positive cells measured 2 days after transduction.

Article Snippet: Recombinant human DSG-2 was from Leinco Technologies (Fenton, MO; catalog no. D340) and recombinant CD46 from Sino Biological (Beijing, China; catalog no. 12239-H08H).

Techniques: Expressing, Flow Cytometry, Plasmid Preparation, In Vitro, Transduction, Infection, Incubation, Recombinant, Staining