Mar 26, 2026
MORF Screen in Primary Human CD8+ T cells
- Christian McRoberts Amador1,2,
- Tomer Rotstein3,2,
- Aretha Gao4,
- Charles Gersbach3,2
- 1Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27708;
- 2Center for Advanced Genomic Technologies, Duke University, Durham, NC 27708;
- 3Department of Biomedical Engineering, Duke University, Durham, NC 27708;
- 4Department of Biology, Duke University, Durham, NC 27708
Protocol Citation: Christian McRoberts Amador, Tomer Rotstein, Aretha Gao, Charles Gersbach 2026. MORF Screen in Primary Human CD8+ T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrbwjplmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 24, 2026
Last Modified: March 26, 2026
Protocol Integer ID: 313858
Keywords: CD8 T cells, T cells, MORF, approach in primary human cd8, morf screen in primary human cd8, primary human cd8, lentiviral overexpression, pooled lentiviral overexpression, transcription factor, downstream multiomic profiling, cd8, systematic identification of regulator
Abstract
We describe a pooled lentiviral overexpression screening approach in primary human CD8+ T cells to identify transcription factors that regulate T cell state under acute and chronic stimulation. CD8+ T cells from multiple donors were isolated, activated, and transduced with a barcoded mORF library, followed by expansion, selection, and stimulation under defined conditions. This protocol enables systematic identification of regulators of T cell dysfunction and state transitions, and can be adapted for targeted sublibrary validation and downstream multiomic profiling.
Guidelines
Blood collection for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee.
Materials
Leukopak - fresh (Stem Cell Technologies - Cat #70500)
CD8+ T cell negative isolation kit (Stem Cell Technologies - Cat #17953)
Dynabeads Human T-Activator CD3/CD28 (Thermo/Gibco - Cat #11132D)
PRIME-XV T cell Expansion XSFM (FujiFilm - Cat #91141-1L)
Human Platelet Lysate (Compass Biomedical - Cat #PLSA-1000)
Penicillin-Streptomycin (Thermo/Gibco - Cat #15140122)
Recombinant Human IL-2 (Thermo/Peprotech - Cat #200-02-1MG)
Lenti-X Concentrator (Takara - Cat #631232)
Puromycin (Invivogen - Cat #ant-pr-1)
eBioscience Cell Stimulation Cocktail (Thermo/Invitrogen - Cat #00-4970-03)
Troubleshooting
Isolation and culture of primary human T cells
Human CD8+ T cells were isolated from three distinct donors (StemCell Tech) using negative selection human CD8 isolation kits (StemCell Tech).
T cells were activated with a 3:1 ratio of anti-CD3/CD28 Dynabeads (Gibco) to T cells.
T cells were cultured in PRIME-XV T cell Expansion XSFM (FujiFilm) supplemented with 5% human platelet lysate (Compass Biomed), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, and 100 U ml-1 of human IL-2 (Peprotech).
T cells were activated with a 3:1 ratio of CD3/CD28 dynabeads to T cells and maintained at 1–2 × 106 cells ml−1 unless otherwise indicated. For chronic stimulation experiments, dynabeads were removed using a magnet, and fresh dynabeads at a 3:1 Dynabead:T cell ratio were added.
Lentivirus generation and transduction of primary human T cells
For all lentivirus generation, a previously described transfection protocol was used to ensure high lentiviral titer (Marson CRISPRi/a cytokine Science paper).
In brief, HEK-293Ts were transfected with lentiviral packaging, envelope, and vector of interest plasmids.
Lentiviral supernatants were harvested 24 hours and 48 hours after transfection, with a media refresh between harvests.
Lentiviral supernatant was centrifuged at 600g for 10 min to remove cellular debris and concentrated to 50–100× the initial concentration using Lenti-X Concentrator (Takara Bio).
T cells were transduced at 5–10% v/v of concentrated lentivirus at 24 h post-activation.
For co-transduction experiments, T cells were transduced at 5-10% v/v of concentrated lentivirus 24 h post activation.
T cell culture
Day 0: CD8+ T cells were transduced (n=3 biological replicates) with the lentiviral library at 10% v/v, 24 hours after isolation and stimulation with anti-CD3-CD8 Dynabeads (see Isolation and culture of primary human T cells), and expanded for 3 days in standard media.
Day 3: Cells were selected with puromycin at 1 µg/mL for two days.
Day 5: Dynabeads were removed, and cells were split into two conditions: acute and chronic. Both conditions were supplemented with 0.5 ug/mL of puromycin, and the chronic condition was additionally restimulated with fresh dynabeads at a 3:1 concentration.
Day 7: both conditions were replenished with standard media with no puromycin. Dynabeads were removed and replenished for the chronic condition at a 3:1 concentration.
Day 10: Acute and chronic conditions were additionally split into two dishes. To one acute sample, and one chronic sample, cells were stimulated were stimulated with 1x T cell stimulation cocktail (Thermo) for two hours. Unstimulated controls were supplemented with an equal volume of ethanol for 2 hours.