biotinylated secondary antibody  (Vector Laboratories)

Journal: iScience

Article Title: Integrating complementary approaches reveals antigen-reactive CD4 + T cell states after SARS-CoV-2 vaccination

doi: 10.1016/j.isci.2026.116175

Figure Lengend Snippet: Identification of SARS-CoV-2-specific T cell responses by reverse phenotyping (A) CoVa-Adapt study design and sample collection scheme. For all donors, PBMCs were collected at day 0 (P0), 10 days after primary (P10), 10 and 210 days after secondary (S10, S210), and 10 and 189 days after tertiary (T10, T189) vaccination. For selected donors, PBMCs were additionally sampled 108 days after tertiary vaccination (T108, n = 7). Vaccination-induced T cell responses were characterized for most donors on a quantitative level by IFNγ ELISpot. Selected CoVa-Adapt donors were subjected to in-depth characterization using scRNAseq (reverse phenotyping and epitope-specific analyses) followed by TCR functional testing. (B–G) scRNAseq data from the reverse phenotyping dataset. For reverse phenotyping, PBMCs were re-stimulated with 15-mer peptides covering the complete wild-type spike protein or left untreated. Sorted non-naïve CD4 + and/or CD8 + T cells were subjected to scRNAseq. Only CD4 + T cells are shown (annotation described in the methods section). The full dataset is depicted in . (B) UMAP of stimulated (blue) and unstimulated (orange) T cells (left) and Leiden clusters (right; cluster names in UMAP, cluster numbers on the right) ( n = 101,939 cells in total). Cells located within the reactive cluster are displayed with increased point size. (C) Dot plots of log-normalized expression of representative genes per cluster. Selected genes of the reactive cluster are highlighted in gray. Numbers on the left indicate cluster numbers with reactive cluster 12 highlighted in bold. (D and E) IFNG expression (D) and proliferation score (E) in unstimulated (stimulated cells in gray) and stimulated (unstimulated cells in gray) CD4 + T cells (left), and quantification in the stimulated condition of cells in the reactive cluster versus all other clusters (right). Cells located within the reactive cluster are displayed with increased point size. For IFNG , cells with log-normalized gene expression of 0 are shown in gray in UMAPs. Statistical testing by the Mann-Whitney U test. (F) UMAP visualization of cells classified as reactive (cells located in the reactive cluster or belonging to clones where at least one cell is in the reactive cluster) from donor A5 at individual time points after primary (P), secondary (S), and tertiary (T) vaccination in the stimulated condition. Color gradient indicates IFNG expression at the indicated time points. Non-reactive cells and cells from other donors are shown in gray. (G) Fraction of cells from donor A5 at each time point belonging to the reactive cluster. (H and I) Identification of spike-reactive T cells after 20h of in vitro re-stimulation of PBMCs with 15-mer peptides covering the complete wild-type spike protein. Peptides were provided in two subpools, S1 (depicted in H) and S2. Primary data (H) of donor A5 is shown. Quantification (I) of spot-forming units (SFU) for IFNγ ELISpot (combined frequencies of S1 and S2 subpools), data points represent individual donors ( n = 12–19 per time point), solid lines indicate the mean. Samples without SFU above the negative control were set to not detected (n.d.). Donor A5 is highlighted in pink. Statistical testing by the Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Significant differences from the P10 time-point are indicated. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, n.s. not significant.

Article Snippet: Plates were washed with PBS containing 0.05% Tween 20 (Sigma-Aldrich, P9416-50 mL) and incubated with biotinylated anti-human IFNγ monoclonal antibody (clone 7-B6-1, Mabtech, 3420-6-250) at 0.2 μg/well for 2 h. Plates were washed a second time with PBS containing 0.05% Tween 20 and subsequently incubated with an avidin-biotinylated peroxidase complex (VECTASTAIN Elite ABC-HRP Kit, Vector Laboratories, VEC-PK-6100) for 1–2 h. Afterward, plates were washed first with PBS containing 0.05% Tween 20 following one washing step with PBS.

Techniques: Enzyme-linked Immunospot, Functional Assay, Expressing, Gene Expression, MANN-WHITNEY, Clone Assay, In Vitro, Negative Control

Journal: Molecular Therapy Oncology

Article Title: Induced pluripotent stem cell-derived dendritic cells potentiate the efficacy of interleukin-12-expressing oncolytic HSV-1

doi: 10.1016/j.omton.2026.201252

Figure Lengend Snippet: Immunological assessments of the combination of T-mfIL12 and immature BMDCs (A) Immunohistochemical analysis of representative MB49 tumors from the indicated treatment groups. Increased intratumoral infiltration of CD8 + lymphocytes was most prominent in the T-mfIL12+BMDC group, followed by the T-01+BMDC group. In both groups, elevated levels of CD4 + lymphocyte infiltration were also observed in the same tumor sections. Other groups, except for the mock group, showed moderate levels of CD4 + infiltration. Scale bars: 100 μm. (B) ELISpot assay for IFN-γ expression revealed the highest number of IFN-γ-producing splenocytes in the T-mfIL12+BMDC group, followed by the T-01+BMDC group. Monotherapies with BMDC, T-01, or T-mfIL12, as well as the mock group, did not result in significant increases. n = 3 per group; all samples were analyzed in triplicate. Error bars represent standard deviation (SD). ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Wells were incubated with biotinylated anti-IFN-γ antibodies (R4-6A2; Mabtech AB, cat. #3321-2A) for 2 h at room temperature, followed by a 1-h incubation with the streptavidin-alkaline phosphatase conjugate.

Techniques: Immunohistochemical staining, Enzyme-linked Immunospot, Expressing, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: Induced pluripotent stem cell-derived dendritic cells potentiate the efficacy of interleukin-12-expressing oncolytic HSV-1

doi: 10.1016/j.omton.2026.201252

Figure Lengend Snippet: Immunological assessments of the combination of T-mfIL12 and immature iPSDCs (A) Immunohistochemical analysis of representative MB49 tumors from the indicated treatment groups. Increased intratumoral infiltration of CD8 + lymphocytes was observed in both the T-mfIL12+BMDC and T-mfIL12+iPSDC groups. In both groups, CD4 + lymphocyte infiltration was also more prominently observed in the same tumor sections. Other treatment groups, excluding the mock group, exhibited moderate levels of CD4 + T cell infiltration. Scale bars: 100 μm. (B) ELISpot assay showed a high and comparable increase in IFN-γ-producing splenocytes in both the T-mfIL12+BMDC and T-mfIL12+iPSDC groups. n = 3 mice per group; all samples were analyzed in triplicate. Error bars represent standard deviation (SD). ∗∗∗∗ p < 0.0001; NS, not significant.

Article Snippet: Wells were incubated with biotinylated anti-IFN-γ antibodies (R4-6A2; Mabtech AB, cat. #3321-2A) for 2 h at room temperature, followed by a 1-h incubation with the streptavidin-alkaline phosphatase conjugate.

Techniques: Immunohistochemical staining, Enzyme-linked Immunospot, Standard Deviation