cd4viobright v600  (Miltenyi Biotec)

Journal: Nature Medicine

Article Title: Multi-antigen-targeting T cells in pediatric central nervous system tumors: a phase 1 trial

doi: 10.1038/s41591-026-04449-9

Figure Lengend Snippet: Flow immunophenotyping release testing data are presented in A-G and IFNγ ELISpot data presented in H-K (Arm A: n = 12; Arms B/C: n = 27). Each data point represents a separate TAA-T cell product infused into patients (biological replicates). Lower whisker is defined as the minima, upper whisker is defined as the maxima, center is defined as median, lower bound of box is defined as Q1 (25th percentile), and upper bound of box is defined as Q3 (75th percentile). a ) % Total T cells (CD3 + ). Arm A: Minima=51, Maxima=99, Median=97, 25th Percentile=95, 75th Percentile=99; Arms B/C: Minima=31, Maxima=100, Median=97, 25th Percentile=94, 75th Percentile=98. b ) % CD4 + T cells (CD3 + CD4+ of CD45 + ). Arm A: Minima=0.50, Maxima=88, Median=16, 25th Percentile=2.6, 75th Percentile=38; Arms B/C: Minima=0.40, Maxima=55, Median=9.9, 25th Percentile=6.1, 75th Percentile=17. c ) %CD8 + T cells (% CD3 + CD8+ of CD45 + ). Arm A: Minima=4.3, Maxima=88, Median=37, 25th Percentile=22, 75th Percentile=47; Arms B/C: Minima=19, Maxima=91, Median=51, 25th Percentile=36, 75th Percentile=71. d ) % αβ T cells (% TCRαβ+ of CD3 + ). Arm A: Minima=8.2, Maxima=94, Median=69, 25th Percentile=28, 75th Percentile=86; Arms B/C: Minima=32, Maxima=98, Median=71, 25th Percentile=48, 75th Percentile=84. e ) % γδ T cells (% TCRγδ+ of CD3 + ). Arm A: Minima=5.1, Maxima=79, Median=28, 25th Percentile=8.4, 75th Percentile=67; Arms B/C: Minima=1.1, Maxima=83, Median=25, 25th Percentile=10, 75th Percentile=53. f ) % CD3 + CD16 + CD56+ of CD45 + . Arm A: Minima=2.8, Maxima=25, Median=11, 25th Percentile=7.2, 75th Percentile=17; Arms B/C: Minima=3.4, Maxima=64, Median=16, 25th Percentile=8.1, 75th Percentile=28. g ) % NK cells (CD16/CD56 + CD3- of CD45 + ). Arm A: Minima=0.40, Maxima=48, Median=1.0, 25th Percentile=0.48, 75th Percentile=4.6; Arms B/C: Minima=0.10, Maxima=62, Median=1.6, 25th Percentile=0.60, 75th Percentile=6.1. h - i ) IFNγ ELISpot with background correction for Arm A ( h ) and Arms B/C ( i ). Negative values after background normalization have been normalized to zero for visual representation.

Article Snippet: An FcR blocking agent (Miltenyi Biotec, cat. no. 130-059-901) was added to the tubes for 5 min to prevent any nonspecific binding, followed by an appropriate antibody mix (shown in tables below), which was incubated in the dark for 15 min. Antibodies for the pan leukocyte panel included CD14 Vio Blue (Miltenyi Biotec, cat. no. 130-110-524), CD19 FITC (Miltenyi Biotec, cat. no. 130-113-646), CD16 PE (Miltenyi Biotec, cat. no. 130-113-393), CD56 PE (Miltenyi Biotec, cat. no. 130-113-312), CD3 Per CP Vio-770 (Miltenyi Biotec, cat. no. 130-113-141), CD4 PE-Vio-770 (Miltenyi Biotec, cat. no. 130-113-227), CD45 APC (Miltenyi Biotec, cat. no. 130-110-633) and CD8 APC Vio-770 (Miltenyi Biotec, cat. no. 130-110-681).

Techniques: Enzyme-linked Immunospot, Whisker Assay

Journal: Molecular Therapy Oncology

Article Title: Boosting CAR T cell functionality with oncolytic viruses for the treatment of pediatric diffuse midline gliomas

doi: 10.1016/j.omton.2026.201191

Figure Lengend Snippet: Phenotypic changes in CD4 and CD8 CAR T cells drive successful combination responses (A) Dot plot demonstrating the log2 (fold change) of markers associated with T cell activation, proliferation, exhaustion, migration, cytotoxicity, memory/naivety, and regulation, expressed across CD4 and CD8 CAR T cell populations in Goravir Non-Responders and Responder conditions and in R124 Non-Responder and Responder conditions. (B) Dotted bar plots showing population percentages of IFN-γ+ CAR T cells from the total population of viable CD3+CD45+CD4+ or viable CD3+CD45+CD8+ cells co-cultured with Goravir- or R124 pre-infected DMGs. (C) Bar plots showing population percentage comparisons of naive T cells (CCR7+CD45RA+), central memory T cells (TCM) (CCR7+CD45RA-), effector memory T cells (TEM) (CCR7-CD45RA-), and terminally differentiated effector memory T cells (TEMRA) (CCR7-CD45RA+) in CD4 (left) and CD8 (right) co-cultured with Goravir (top) or R124 (bottom) pre-infected DMGs. For the dot plot, data are presented as mean log2 (fold change) measured from B7H3 ( n = 6) and GD2 ( n = 6)-co-cultures of DMG (SU-DIPG-IV, VUMC-DIPG-G, JHH-DIPG-01, HSJD-DIPG-07, VUMC-DIPG-10, and VUMC-DIPG-11) per condition (uninfected, Goravir pre-infected, R124 pre-infected), indicated as decreasing log2 (fold change) (blue), increasing (red), and unaffected (white), using expression in CAR T cells co-cultured with each uninfected DMG as baseline. The size of each dot plot indicates −log10 (adjusted p value), with increasing sizes indicating significant differences compared with the uninfected condition. Additional lines and asterisks were used to indicate significant differences between Responders and Non-Responders. Each box and bar in the bar plots represents mean ± SD of specific population % from CAR T cell co-cultures, with each point color-coded to represent the population % of B7H3 (square) ( n = 18) or GD2 (circle) ( n = 18) CAR T cells across uninfected, Non-Responders, and Responder conditions of either Goravir or R124 pre-infected DMG co-cultures. Statistical analysis was performed using repeated-measures one-way ANOVA corrected with Tukey’s test for parametric data, while for non-parametric data, the Friedman paired test corrected with Dunn’s test for multiple corrections was used. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. The dot plot was plotted in R ( https://www.r-project.org/ ).

Article Snippet: The CD4+ and CD8+ proportions, as well as CAR expression, were evaluated using fluorescence-activated cell sorting (FACS) after expansion, using CD3-APC (1:100, Biolegend, #300412), CD4 Violet Blue (1:50, Miltenyi biotec, #130-113-219), and CD8-V500 (1:50, BD, #561617), B7H3-CAR T cells were detected using a B7H3 detection reagent (ACRO Biosystems, B7B-HF2E7,DE, USA), and GD2-CAR T cells were detected using fluorescein isothiocyanate (FITC)-conjugated anti murine IgG (Sigma-Aldrich, #F9137, Amsterdam, the Netherlands).

Techniques: Activation Assay, Migration, Cell Culture, Infection, Expressing

Journal: bioRxiv

Article Title: Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

doi: 10.64898/2026.06.11.725753

Figure Lengend Snippet: ( A ) SupT1 R5 donor cells infected with the HIV-1 molecular clone NL4-3 were co-cultured with uninfected SupT1 R5 target cells for 3 hours at 37°C. Cells were fixed, permeabilized, and immunostained for HIV-1 Env (yellow), CD4 (red), and p24/Gag (green), with nuclei counterstained using Hoechst 33342 (blue). ( B ) Percentage of newly infected target SupT1 R5 cells after 24 hours. NL4-3 IRES-eGFP infected SupT1 R5 cells (GFP+) were either co-cultured with CellTracker Deep Red stained target SupT1 R5 cells (CTDR+) or within a transwell system where infected and target cells were separated by a 0.4 µm membrane to block cell-cell contact. Data is representative of two biological replicates. Black bar represents the mean and the gray box represents standard deviation, which are also given below the x-axis. p -values calculated using two-tailed unpaired t- test.

Article Snippet: The cells were incubated overnight with the broadly neutralizing anti-Env antibody 2G12 (BEI Resources, #ARP-1476) and with conjugated antibodies for detection of p24 (KC57-FITC Conjugate) (BEI Resources, #ARP-13450) and human CD4 (CD4-APC Conjugate) (Miltenyi #130-113-222) at 1:200 dilution in a solution of PBS, 0.05% saponin, 1% BSA and 2% normal goat serum.

Techniques: Infection, Cell Culture, Staining, Membrane, Blocking Assay, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

doi: 10.64898/2026.06.11.725753

Figure Lengend Snippet: ( A ) Representative dot plots of multicell conjugates in 3h co-cultures between target CTDR+ SupT1 R5 cells mixed with either NL4-3 IRES-eGFP or ΔEnv IRES-eGFP infected SupT1 R5 cells. ( B ) The percentage of GFP+CTDR+ multicell conjugate events from initial 0-hour or 3-hour co-cultures. ( C ) Representative dot plots of single cells in 24-hour co-cultures of target CTDR+ SupT1 R5 cells mixed with either NL4-3 IRES-eGFP or ΔEnv IRES-eGFP+ infected SupT1 R5 cells ( D ) The percentage of GFP+CTDR+ single cell events from initial 0-hour or 24-hour co-cultures. Distinct shapes in B and D each represent distinct biological replicates (N = 3) and each individual shape a technical replicate. (E-H) Same as A-D but with co-culture of infected GFP+ primary CD4+ T lymphocytes and target CTDR+ primary CD4+ T lymphocytes. Shapes in F and H represent distinct primary cell donors (N = 4). All values are normalized to initial GFP+ infection levels prior to co-culture. Values below x-axis indicate the mean and standard deviation for each category. Black bar represents the mean and the gray box represents ±1 standard deviation. P-values calculated using either two-tailed unpaired t- test ( B and D ) or two-tailed paired t- test ( F and H ).

Article Snippet: The cells were incubated overnight with the broadly neutralizing anti-Env antibody 2G12 (BEI Resources, #ARP-1476) and with conjugated antibodies for detection of p24 (KC57-FITC Conjugate) (BEI Resources, #ARP-13450) and human CD4 (CD4-APC Conjugate) (Miltenyi #130-113-222) at 1:200 dilution in a solution of PBS, 0.05% saponin, 1% BSA and 2% normal goat serum.

Techniques: Infection, Single Cell, Co-Culture Assay, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

doi: 10.64898/2026.06.11.725753

Figure Lengend Snippet: ( A , D ) Quantification of GFP+CTDR+ multicell conjugates events in 3-hour co-cultures of target CTDR+ SupT1 R5 and NL4-3 IRES-eGFP infected SupT1 R5 cells treated with varying concentrations of soluble CD4 (sCD4, A ) or anti-CD4 (SIM.2, D ). ( B and E ) Quantification of GFP+CTDR+ single cells in 24-hour co-cultures treated with varying concentrations of sCD4 ( B ) or SIM.2 ( E ). ( C and F ) Quantification of GFP+ SupT1 R5 after cell-free NL4-3 IRES-eGFP virus infection treated with varying concentrations of sCD4 ( C ) or SIM.2 ( F ). Data are derived from distinct biological replicates (N = 2), where colors represent distinct biological and each dot a technical replicate. All values are normalized to untreated controls and initial GFP+ infection levels prior to co-culture (if applicable). Values below x-axis indicate the mean and standard deviation for each category. Black bar represents the mean and the gray box represents ±1 standard deviation. P-values calculated using one-way ANOVA with a Tukey’s test for multiple comparisons.

Article Snippet: The cells were incubated overnight with the broadly neutralizing anti-Env antibody 2G12 (BEI Resources, #ARP-1476) and with conjugated antibodies for detection of p24 (KC57-FITC Conjugate) (BEI Resources, #ARP-13450) and human CD4 (CD4-APC Conjugate) (Miltenyi #130-113-222) at 1:200 dilution in a solution of PBS, 0.05% saponin, 1% BSA and 2% normal goat serum.

Techniques: Infection, Virus, Derivative Assay, Co-Culture Assay, Standard Deviation