cd8 media  (R&D Systems)

Journal: bioRxiv

Article Title: Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8 + T cells

doi: 10.64898/2026.04.28.721204

Figure Lengend Snippet: Surface MHC levels on uninfected or HIV-1-infected primary CD4 + T cells. Mean fluorescence intensity (MFI) of untreated (NT) or PHA-activated (GFP - , GFP + ) primary human CD4 + T cells. Activated cells were infected with the HIV-1 reporter construct NL4-3 Δenv -eGFP and sorted into a GFP-negative and GFP-positive population. Cells were stained with lineage and viability markers and with PE-conjugated anti-human HLA-A2 (A) or PE-conjugated anti-human HLA-E (B), and the MFI of the viable CD3 + CD8 - population was measured by flow cytometry. Each dot on the bar plot represents the average MFI for one HIV-negative donor (n = 4). Normalized histograms from one representative experiment are shown on the right. Statistical significance was determined through unpaired two-tailed t-test; ns: no significance (p-value > 0.05); **: p-value < 0.005; ***: p-value <0.0005.

Article Snippet: PBMCs from HIV-negative donors were stimulated with soluble anti-CD3 monoclonal antibodies (BioLegend, clone OKT3, 15ng/mL, Cat. # 317302) for 72 hours in CD8 media (RPMI1640 (FisherScientific)+ 10% Fetal Bovine Serum (GeminiBio) + 1% Penicillin/Streptomycin (ThermoFisher) + 100 U/mL IL-2 (R&D Systems) + 5 ng/mL hrIL-7 (BioLegend)).

Techniques: Infection, Fluorescence, Construct, Staining, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8 + T cells

doi: 10.64898/2026.04.28.721204

Figure Lengend Snippet: Impact of single residue substitution in Mtb44 on the interactions with HLA-E and RLP-13. K562-3.3 Cells were pulsed with a library of 171 variant peptides where each residue (rows) was substituted with each other possible amino acid (columns) for 4 hours in serum-free media. (A) Cells were stained with PE-conjugated anti-HLA-E monoclonal antibody and surface levels of HLA-E were assessed through flow cytometry. (B) PBMCs were stimulated with anti-human CD3 monoclonal antibody and CD8-positive T cells were isolated and cultured without activating stimuli for 7 days. Pulsed cells from (A) were co-cultured with CD8 + T cells and RLP-13. The concentration of chemokine MIP-1β in the co-culture supernatant was quantified through ELISA. The framed cells in the heatmaps correspond do the amino acid sequence in the original Mtb44 peptide.

Article Snippet: PBMCs from HIV-negative donors were stimulated with soluble anti-CD3 monoclonal antibodies (BioLegend, clone OKT3, 15ng/mL, Cat. # 317302) for 72 hours in CD8 media (RPMI1640 (FisherScientific)+ 10% Fetal Bovine Serum (GeminiBio) + 1% Penicillin/Streptomycin (ThermoFisher) + 100 U/mL IL-2 (R&D Systems) + 5 ng/mL hrIL-7 (BioLegend)).

Techniques: Residue, Variant Assay, Staining, Flow Cytometry, Isolation, Cell Culture, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: bioRxiv

Article Title: Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8 + T cells

doi: 10.64898/2026.04.28.721204

Figure Lengend Snippet: Specificity of RLP-13 in dual-color co-cultures. (A) Setup of dual-color co-cultures: K562 cells transfected with covalently linked peptide:HLA-E expression constructs, presenting either cognate (Mtb44) or non-cognate (SP-2A) peptide, are stained with CellTrace Violet (CTV) or CFSE, respectively, and cultured for 18 hours with pre-expanded CD8 + T cells and different concentrations of RLP-13 at a ratio of 4:1:3 (Effector : Cognate target : Non-cognate target). Cells are then stained with lineage and activation markers and viability dye for analysis by flow cytometry. (B) Representative flow plot of viable single cell populations from co-culture wells without (left) or with (right) RLP-13. Indicated cognate and non-cognate gated population frequencies were used to calculate the relative viability of target cell populations. (C) Target cell viability of indicated cognate and non-cognate populations from co-cultures with various concentrations of either RLP-13 (red and pink lines) or irrelevant control scDb H2-mu (black and green lines). Viability was calculated by normalizing the frequency of the indicated target cell populations to that observed in co-culture wells without any scDb added. Assay was repeated five independent times across two HIV-negative donors. Shown measurements are averaged over eight technical replicates from one representative experiment. (D) Concentrations of the indicated effector molecules in the supernatants of co-cultures shown in (C) measured using the LegendPlex CD8/NK cell cytokine panel kit.

Article Snippet: PBMCs from HIV-negative donors were stimulated with soluble anti-CD3 monoclonal antibodies (BioLegend, clone OKT3, 15ng/mL, Cat. # 317302) for 72 hours in CD8 media (RPMI1640 (FisherScientific)+ 10% Fetal Bovine Serum (GeminiBio) + 1% Penicillin/Streptomycin (ThermoFisher) + 100 U/mL IL-2 (R&D Systems) + 5 ng/mL hrIL-7 (BioLegend)).

Techniques: Transfection, Expressing, Construct, Staining, Cell Culture, Activation Assay, Flow Cytometry, Single Cell, Co-Culture Assay, Control

Journal: bioRxiv

Article Title: Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8 + T cells

doi: 10.64898/2026.04.28.721204

Figure Lengend Snippet: Quantification of additional secreted cytokines, chemokines and effector molecules in co-culture supernatant from . Quantification performed using the BioLegend LEGENDPlex TM Human CD8/NK Panel (13-plex) kit following manufacturer protocol. ULOQ, upper limit of quantification. Black symbols: supernatant from co-culture with irrelevant control scDb; red symbols: co-culture with RLP-13. Empty symbols: values outside of quantifiable range (either below limit of detection or above ULOQ).

Article Snippet: PBMCs from HIV-negative donors were stimulated with soluble anti-CD3 monoclonal antibodies (BioLegend, clone OKT3, 15ng/mL, Cat. # 317302) for 72 hours in CD8 media (RPMI1640 (FisherScientific)+ 10% Fetal Bovine Serum (GeminiBio) + 1% Penicillin/Streptomycin (ThermoFisher) + 100 U/mL IL-2 (R&D Systems) + 5 ng/mL hrIL-7 (BioLegend)).

Techniques: Co-Culture Assay, Control

Journal: bioRxiv

Article Title: Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8 + T cells

doi: 10.64898/2026.04.28.721204

Figure Lengend Snippet: Efficacy and sensitivity of RLP-13 in primary cell co-cultures for different HLA-E alleles. (A) Overview of co-culture setup: PBMCs isolated from HIV-negative donors homozygous for either HLA-E*01:01 or HLA-E*01:03 were pulsed with various concentrations of Mtb44-peptide, and cultured with pre-expanded autologous CD8 + T cells and either 0 nM or 2 nM RLP-13 overnight at an effector-to-target ratio of 3:1. Cells were then stained with lineage and activation markers and viability dye. (B) Viability curves of target cells from co-cultures described in (A). Assay was performed four times across two donors. Shown measurements are averaged over at least six technical replicates from representative assays for each donor. (C) and (D) show effector molecule concentrations in supernatants from co-cultures in (B) measured using the LegendPlex CD8/NK cell cytokine panel kit.

Article Snippet: PBMCs from HIV-negative donors were stimulated with soluble anti-CD3 monoclonal antibodies (BioLegend, clone OKT3, 15ng/mL, Cat. # 317302) for 72 hours in CD8 media (RPMI1640 (FisherScientific)+ 10% Fetal Bovine Serum (GeminiBio) + 1% Penicillin/Streptomycin (ThermoFisher) + 100 U/mL IL-2 (R&D Systems) + 5 ng/mL hrIL-7 (BioLegend)).

Techniques: Co-Culture Assay, Isolation, Cell Culture, Staining, Activation Assay

Journal: bioRxiv

Article Title: Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8 + T cells

doi: 10.64898/2026.04.28.721204

Figure Lengend Snippet: RLP-13 induces antigen-dependent lysis of cells expressing HIV-1 construct encoding for Mtb44 peptide (A) Outline of the NL4-3 Δenv -Mtb44 reporter construct. The provirus encodes for all HIV-1 gene products except env , which is truncated by an inserted eGFP reporter-cassette followed by a non-coding T2A-sequence and the DNA sequence encoding for the Mtb44-peptide. (B) Experiment setup: PBMCs from isolated from three HIV-negative donors. PBMCs were treated with PHA for 72 hours before infection with the indicated HIV-1 reporter construct without (ΔEnv) or with (ΔEnv-Mtb44) the insert encoding for the Mtb44 -peptide. Effector cells were prepared by culturing autologous PBMCs with anti-human CD3 monoclonal antibody, and CD8 + T cells were isolated and rested for 7 days. Target and effector cells were co-cultured overnight at a ratio of 1:1 with varying concentrations of RLP-13. Cells were then stained for viability, lineage and activation markers and analyzed by flow cytometry. (C) Selected flow cytometry plots from a representative experiment as described in (B). Target cells were identified as CD3 + CD8 - single cells. Each plot shows data from one well containing target cells infected with the indicated virus for each column and cultured with autologous CD8 + T cells and the indicated scDb for each row. (D) Frequency of viable GFP-positive (left) or GFP-negative target cells (right). PHA-treated, activated CD4 + T cells were infected with either NL4-3 Δenv -eGFP or NL4-3 Δenv -Mtb44 and cultured with autologous, pre-expanded CD8 + T cells and indicated concentrations of RLP-13. Target cells were identified as CD3 + CD8 - single cells. GFP-expressing indicates active expression of integrated provirus. Viability was normalized to an irrelevant scDb (H2-mu) control. Measurements are averaged over four technical replicates. Two independent repeat experiments were performed for three HIV-negative donors; data shown is from one representative experiment. (E) Concentrations of the indicated effector molecules in the supernatant of co-cultures from (D).

Article Snippet: PBMCs from HIV-negative donors were stimulated with soluble anti-CD3 monoclonal antibodies (BioLegend, clone OKT3, 15ng/mL, Cat. # 317302) for 72 hours in CD8 media (RPMI1640 (FisherScientific)+ 10% Fetal Bovine Serum (GeminiBio) + 1% Penicillin/Streptomycin (ThermoFisher) + 100 U/mL IL-2 (R&D Systems) + 5 ng/mL hrIL-7 (BioLegend)).

Techniques: Lysis, Expressing, Construct, Sequencing, Isolation, Infection, Cell Culture, Staining, Activation Assay, Flow Cytometry, Virus, Control

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Experimental method to assess resistance to CTL-mediated killing. (A) Co-culture setup: primary CD4 + T cells from PLWH are pulsed with mutant p53 peptide and cultured with autologous, pre-expanded CD8 + T cells and p53-specific scDb. IPDA is performed on the viable CD4 + T cell population isolated before and after the co-culture to assess for enrichment of infected cells, indicative of resistance. (B) scDb mechanism of action: scDbs bind to cognate peptide:MHC complexes on the target cell surface and also to the CD3ε subunit of the TCR complex to induce localized cytotoxicity. (C) Flow cytometry gating strategy to determine frequency of viable target cells. (D) Representative histograms of co-cultures with and without scDbs. The reduction in the viable CD4 + T cell count (marked region) was used to determine the amount of CTL-mediated lysis. I Representative viability data for study participant OM5418 for the experimental condition and seven control conditions.

Article Snippet: Effector cells for co-cultures were expanded through stimulation with soluble anti-CD3 mAb (15 ng/mL, clone OKT3, BioLegend, Cat. # 317302) in CD8 media (base media + 250 U/uL human IL-2 (R&D Systems, Cat. # 202-IL-050/CF) + 5 ng/mL recombinant human IL-7 (BioLegend, Cat. # 581906)) for 72 hours.

Techniques: Co-Culture Assay, Mutagenesis, Cell Culture, Isolation, Infection, Flow Cytometry, Cell Characterization, Lysis, Control

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Effect of active viral expression on cell death resistance. (A) Experimental setup: Healthy donor CD4 + T cells were activated for 72 hours with PHA and infected with different HIV-1 GFP-reporter constructs. Cells were then pulsed with exogenous p53 R175H peptide and co-cultured with pre-expanded autologous CD8 + T cells and p53-specific scDb or irrelevant control scDb. Viability and GFP-expression were analyzed through flow cytometry. (B) Genome maps of the HIV-1 reporter constructs used to infect target cells. Each row represents one reading frame. Striped segments indicate gene segments that are no longer translated due to insertion of a STOP-codon. (C) Representative flow plot showing viable uninfected (GFP-negative) and HIV-1-expressing (GFP-positive) cell populations. (D) Viability of uninfected target cells or cells infected with indicated HIV-1 reporter constructs after co-culture with the HLA-A2:p53 R175H -restricted scDb. Each dot shows the average of three technical replicates per condition. Symbols represent individual donors; assay was performed 2 independent times for each of the n=5 healthy donors, except for the full-length condition, where one donor was only tested once for technical reasons. A p-value less than 0.05 was considered significant. Abbreviations: PHA, phytohemagglutinin; LTR, long terminal repeat; IRES, internal ribosome entry site. ns: no significance (p > 0.05).

Article Snippet: Effector cells for co-cultures were expanded through stimulation with soluble anti-CD3 mAb (15 ng/mL, clone OKT3, BioLegend, Cat. # 317302) in CD8 media (base media + 250 U/uL human IL-2 (R&D Systems, Cat. # 202-IL-050/CF) + 5 ng/mL recombinant human IL-7 (BioLegend, Cat. # 581906)) for 72 hours.

Techniques: Expressing, Infection, Construct, Cell Culture, Control, Flow Cytometry, Co-Culture Assay