Journal: bioRxiv

Article Title: Loss of Vpr-driven TRAIL-R2 expression protects HIV-infected cells from non-canonical NK cell TRAIL attack

doi: 10.64898/2026.04.15.718741

Figure Lengend Snippet: (A) Volcano plot of DEGs for the sorted infected populations with vs without NK cell exposure. Black dots indicate a p-adjusted value less than 0.05. Highlighted in orange are all the genes identified in the Hallmark “TNFA signaling via NF-κB” pathway. Highlighted in blue are all the genes identified in the Hallmark “Interferon gamma response” and “Interferon alpha response” pathways. See also Fig S6A for the expanded GSEA Hallmark analysis. (B and C) Detection of cytokines in co-culture. (C) NK cell expression of TNF-α and IFN-γ detected in mock vs infected cell co-cultures. See also Fig S6B and C for gating strategies. Summary data of TNF-𝘢 expression from 9 independent experiments (n=14 donors). Summary data of IFN-ɣ expression from 3 independent experiments (n=6 donors). (C) Detection of type I IFN via BST-2 surface expression on CD4 + T cells in cultures treated with recombinant IFN-β or in NK cell co-cultures. See also Fig S6D and E. Summary data from 3 independent experiments (n=6 donors). Statistical analysis for B and C: paired t test, **p<0.01, ***p<0.001, ****p<0.0001. (D) Cytokine-induced and NK cell co-culture changes in MHC-I on infected cells. Infected cultures were cultured overnight +/- 1µg/mL recombinant cytokines (TNF-𝘢, IFN-ɣ, or IFN-β) or +/- NK cells, followed by measurement of changes in HLA-A*02, HLA-B, HLA-C, and HLA-E gMFI by flow cytometry. Shown are the infected cell average fold changes for each condition from 6 independent cytokine experiments (n=12 donors) and 7 independent co-culture experiments (n=15 donors). Statistical analysis: one sample t test comparison to 1.0, *p<0.05, ***p<0.001, and ****p<0.0001. See also Fig S6F for JR-CSF and REJO.c experiments. (E-G) Effects of TNF-𝘢 pathway manipulation on HLA expression and killing. (E and F) The TNFRSF1A gene was disrupted using CRISPR-Cas9 to ablate TNFR1 expression on CD4 + T cells, followed by HIV infection and overnight cultures with either 1µg/mL recombinant TNF-𝘢 or NK cells (E:T 1). A non-targeting guide RNA was used as a control. Shown are data from 5 independent experiments (n=6 donors). (E) Fold changes in HLA-A*02 and HLA-B expression were measured via flow cytometry. Statistical analysis: multiple paired t tests, **p<0.01 and ***p<0.001. See also Fig S6G and H for IFNGR1 and IFNAR1 knockouts, respectively. (F) NK cell killing of control vs TNFR1 KO infected cells was assessed via flow cytometry. Statistical analysis: paired t test. (E) HIV 89.6 -infected cells were pre-treated overnight with 1µg/mL recombinant TNF-𝘢, followed by overnight co-culture with NK cells (E:T 1) to assess killing. Shown are data from 4 independent experiments (n=8 individual donors). Statistical analysis: paired t test.

Article Snippet: Recombinant cytokines were spiked in at final concentrations of 1μg/mL per cytokine: TNF-α (Biolegend, Cat.#570104), IFN-γ (Biolegend, Cat.#713906), and IFN-β (R&D Systems, Cat.#8499-IF/CF).

Techniques: Infection, Co-Culture Assay, Expressing, Recombinant, Cell Culture, Flow Cytometry, Comparison, CRISPR, Control