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: Potential mechanisms for infected cell resistance to CTL-mediated lysis. CD4 + T cells exhibit stable differences in susceptibility to lysis (A and B). If resistance is associated with properties that allow preferential infection, then infected cells may show greater resistance than uninfected cells (A). Alternatively, intrinsically susceptible cells may be eliminated over time, with death-resistant HIV-1-harboring cells remaining (B). A third possibility (C) is that HIV-1 induces phenotypic changes that increase cell death resistance of infected cells (HIV-induced resistance).

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Infection, Lysis

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: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

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: Ex vivo co-cultures with cells from PLWH do not show enrichment for infected cells in 10/12 participants. (A) Representative ddPCR plots of IPDA performed on CD4 + T cells from donor CIRC0226 before and after co-culture. Each dot represents one droplet out of at least 10,000 droplets measured per assay. Measured frequencies were corrected for cell count and DNA shearing as described previously . (B) Pre- and post-culture frequency of intact provirus per 1x10 6 CD4 + T cells. Each point represents the calculated frequency of intact provirus for one assay for the indicated study participant. (C) Violin plot of enrichment for total study cohort. Each dot represents the average enrichment score for an individual study participant. Colors and symbols coding as in (B). Dashed line marks no change in frequency; horizontal lines mark the median and first and third quartile. (D) Correlation between extent of killing and enrichment for intact proviruses. (E) Correlation between time on ART and enrichment of intact provirus. (F) Comparison of enrichment of intact provirus between study participants with less than 10 years of continuous ART (short-term) and over 13 years of continuous ART (long-term).

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Ex Vivo, Infection, Co-Culture Assay, Cell Characterization, Comparison

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: pMHC-specific latency reversal by the p53-specific scDb. (A) Frequencies of CD69 + CD4 + T cells in individual co-cultures without and with scDb across at least two co-cultures for all twelve study participants. Co-cultures were set up as described in . Error bars represent standard deviation. (B) Induction of viral RNA by the p53-specific scDb. Primary CD4 + T cells from HIV-1-positive study participants were pulsed with the p53 R175H peptide (n = 3) or an irrelevant control peptide (HLA-A2 signal peptide, VMAPRTLVL; n = 1) and cultured for 18 hours with or without p53-specific scDb. RNA was isolated and reverse transcribed. Digital PCR was performed with probes binding to poly-adenylated HIV-1 RNA (poly-A), the integrase region of HIV-1 pol (int) or the splice junction for vpu-env mRNA. (C-E) pMHC-specific increase in HIV-1 transcripts induced by the p53-specific scDb. Measurements were averaged over two technical replicates for each of three identical culture wells per condition, and normalized to transcript level in the no treatment (NT) control. PMA/ionomycin was included as positive control. Colors represent treatment conditions: black: no treatment; red: p53-specific scDb; green: PMA/I. NC: non-cognate peptide (HLA-A2 signal peptide SP-2A, VMAPRTLVL), C: cognate peptide (p53 R175H , HMTEVVRHC).

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Standard Deviation, Control, Cell Culture, Isolation, Reverse Transcription, Digital PCR, Binding Assay, Positive 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: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

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

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: Modified assay targeting stably expressed HLA-E. (A) Cell surface levels of classical HLA-A2 and non-classical HLA-E molecules on untreated, uninfected and HIV-1-expressing cells. Healthy donor CD4 + T cells were either left untreated (NT) or activated with PHA over 72 hours and infected with indicated reporter virus construct. Cells were then sorted by GFP-expression and stained with PE-labeled monoclonal antibodies for HLA-A2 or HLA-E. Each bar represents average measurements across n=3 healthy donors. (B) Viability of uninfected target cells or cells infected with indicated HIV-1 reporter constructs after co-culture with the HLA-E-restricted RLP-13 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. Bars indicate average viability and standard deviation.

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Modification, Stable Transfection, Expressing, Infection, Virus, Construct, Staining, Labeling, Bioprocessing, Co-Culture Assay, Standard Deviation

Journal: eLife

Article Title: CARD8 inflammasome activation during HIV-1 cell-to-cell transmission

doi: 10.7554/eLife.102676

Figure Lengend Snippet: ( A ) CD4+ T cells from a blood donor were isolated, activated, and either mock-infected or infected with HIV-1 NL4.3-BaL for 3 days such that ~10% of cells were positive for intracellular p24 gag . Monocyte-derived macrophages (MDMs) were primed with Pam3CSK4 then cocultured with mock- or HIV-1-infected primary CD4 T cells in the presence or absence of lopinavir (LPV), nevirapine (NVP), or VX765, inhibiting HIV protease, reverse transcriptase, or caspase 1, respectively. Supernatants were harvested 3 days post coculture to assay for IL-1β secretion via IL-1 reporter assay. ( B ) CD4+ T cells from donor 12 and MDMs from donor 14 were cocultured as in (A) in the presence or absence of LPV. Supernatant was harvested at 4, 24 and 48 hours post coculture to probe for IL-1β secretion. ( C ) AAVS1 or CARD8 MDM KOs were immunoblotted using an anti-CARD8 antibody or anti-vinculin. Full-length and FIIND-processed CARD8 intermediates are marked with a purple arrow. Size is indicated in kDa on left side of blot. ( D ) AAVS1 or CARD8 KO MDMs from ( C ) were cocultured with CD4+ T cells infected with HIV-1 NL4.3-BaL then assayed for IL-1β secretion 48 hours post coculture. The donor 12 cocultures consisted of autologous CD4s and MDMs, whereas the MDMs from donors 13–15 were cocultured with donor 12 CD4s. Dotted line indicates limit of detection (LoD). Datasets represent mean ± SD ( A, D : n=2 technical replicates for each donor, B : n=3 technical replicates for one donor). ( A ) Two-way ANOVA with Tukey’s test ( D ) One-way ANOVA with Sidak’s test using GraphPad Prism 10. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 4—source data 1. Original western blot images used to generate . Figure 4—source data 2. Original western blot .tif files used to generate .

Article Snippet: Primary CD4+ T cells were isolated via positive selection using the EasySep Release Human CD4 Positive selection kit (Stem Cell Technologies cat: 17752) according to the manufacturer’s instructions from PBMCs collected from blood donors and seeded at 2.5 × 10 6 cells/mL in the presence of 100 U/mL IL-2.

Techniques: Isolation, Infection, Derivative Assay, Reverse Transcription, Reporter Assay, Western Blot