human ifnɣ hrp  (Mabtech Inc)

Journal: Nature Communications

Article Title: LANA-specific CD4 + effector T cells accumulate at the site of KSHV infection in humanized mice

doi: 10.1038/s41467-025-66992-2

Figure Lengend Snippet: A Schematic of primary TCR-transduced T cell generation for downstream assays. B IFNɣ levels of primary TCR-transduced T cells or normalized luminescence of TCR-transduced Jurkat Lucia NFAT cells co-cultured overnight with HLA-matched LCLs pulsed with decreasing peptide concentrations. Graph shows mean ± SD of n = 2 (Jurkat Cl33), n = 3 (Jurkat Cl156/12, primary Cl114) or n = 4 (rest) independent experiments. C IFNɣ ELISA of TCR-transduced T cells co-cultured with cognate peptide-pulsed or unpulsed HLA-matched LCLs. Boxplots represent 5 (Cl114, Cl33), 4 (Cl12) or 3 (Cl156) independent experiments, using T cells from 8 (Cl114, Cl33, Cl12) or 6 (Cl156) different donors, tested against 9 (Cl114), 6 (Cl33), 4 (Cl156), 3 (Cl12) different LCLs. D Frequency of CD107a + T cells after 6 h of co-culture with cognate peptide-pulsed or unpulsed HLA-matching LCLs. Boxplots represent 3 (Cl114, Cl33, Cl12) or 1 (Cl156) independent experiments, using T cells from 6 (Cl12), 4 (Cl114, Cl33) or 2 (Cl156) different donors, tested against 6 (Cl114), 5 (Cl33), 4 (Cl156) or 3 (Cl12) different LCLs. E Specific killing of HLA-matched pulsed or unpulsed LCLs after co-culture with TCR-transduced T cells. Boxplots represent 3 (Cl156, Cl33, Cl12) or 2 (Cl114) independent experiments, using T cells from 6 (Cl156, Cl33), 5 (Cl12) or 3 (Cl114) different donors, tested against 5 (Cl114, Cl156, Cl33) or 2 (Cl12) different LCLs. C–E Paired two-tailed t-test. Intracellular cytokine staining (6 h) and surface marker assessment (18 h) of CD4 + ( F ) and CD8 + ( G ) T cells after stimulation with PMA/Ionomycin, cognate peptide-pulsed or unpulsed LCLs or medium only as a negative control. Mean frequency of cells positive for the indicated marker (y-axis) in each condition (x-axis) is plotted from 3 (CD4 + ) or 1 (CD8 + ) independent experiments with 5 (CD4 + ) or 2 (CD8 + ) different T cell donors. Numerical means are shown for LCL-stimulated conditions. Asterisks indicate significance from paired two-sided t-tests comparing pulsed versus unpulsed LCLs. A–E * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact significant p ≥ 0.0001 from left to right (2D) 0.00015; (2E) p = 0.00236, p = 0.00019.

Article Snippet: IFNɣ or TNF was measured in the supernatant according to the manufacturer’s instructions (Human IFNɣ (HRP), Mabtech, 3420-1H; Human TNF (HRP), Mabtech, 3512-1H) and the signal was detected on an Infinite 200 PRO (Tecan).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Two Tailed Test, Staining, Marker, Negative Control

Journal: Nature Communications

Article Title: LANA-specific CD4 + effector T cells accumulate at the site of KSHV infection in humanized mice

doi: 10.1038/s41467-025-66992-2

Figure Lengend Snippet: A Schematic of EBV&KSHV co-infected LCL (EK LCL) generation. B Flow cytometry histograms of GFP expression in different LCLs. C Western blot of whole-cell lysates from iSLK.219 and Brk.219 (± lytic induction) and from EBV-only or puromycin-selected EK LCLs (4 donors) stained for LANA and Tubulin. Tubulin-normalized LANA quantification indicated below blots. D IFNɣ production by TCR-transduced T cells co-cultured with HLA-matched LCLs ( ± KSHV infection), normalized to untransduced controls. Boxplots representing five (Cl33), four (Cl114, Cl12) or three (Cl156) independent experiments using T cells from eight (Cl114, Cl33, Cl12) or six (Cl156) donors, tested against six (Cl114, Cl33), four (Cl156) or three (Cl12) LCLs. Paired two-tailed t-test. E Frequencies of proliferating (CTV-diluted) or mouse CD19 + CD8 + T cells after seven-day co-culture of Cl12-transduced CD8 + T cells with irradiated HLA-matched or mismatched LCLs (± KSHV infection, ± peptide-pulsed), normalized to untransduced T cells. Boxplots representing two independent experiments with four T-cell and two target-cell donors. Linear mixed model fit by REML, T cell and LCL donor as a random effect, Holm-adjusted Tukey test. F IFNɣ secretion by T cells co-cultured with peptide-pulsed, KSHV-infected, or untreated MC116 B cells, or medium only. IFNɣ levels are shown relative to PMA/Ionomycin controls. Median ± SD of 2 independent experiments using T cells from three (Cl12) or four (rest) donors. Two-way ANOVA, Tukey’s multiple comparisons test, multiplicity adjusted p -values. G IFNɣ ELISA of TCR-transduced T cells co-cultured with autologous B cells infected with EBV or EBV&KSHV on days 1, 5, 14 post-infection. Mean of one experiment with two donor pairs. H IFNɣ ELISA of untransduced T cells incubated 72 h with conditioned media from freshly infected B cells. Mean ± SD of one experiment with three donor pairs. One-way ANOVA on log 10 -transformed data, Tukey’s multiple comparisons test. ( A–H ) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact significant p ≥ 0.0001 (left to right, top to bottom): (3D) p = 0.03852; (3E) p = 0.00034; (3 F) Cl156: p = 0.0078, p = 0.0013, p = 0.0107; Cl33: p = 0.0224; Cl114: p = 0.0402, p = 0.0472; Cl12: p = 0.0016; (3H) p = 0.0037.

Article Snippet: IFNɣ or TNF was measured in the supernatant according to the manufacturer’s instructions (Human IFNɣ (HRP), Mabtech, 3420-1H; Human TNF (HRP), Mabtech, 3512-1H) and the signal was detected on an Infinite 200 PRO (Tecan).

Techniques: Infection, Flow Cytometry, Expressing, Western Blot, Staining, Cell Culture, Two Tailed Test, Co-Culture Assay, Irradiation, Enzyme-linked Immunosorbent Assay, Incubation, Transformation Assay