Autologous Liver Organoids and T Cells Reveal Donor-Dependent Flucloxacillin Injury

A Direct-Toxicity Assay Was Extended to Adaptive Immune Injury

An Advanced Science study led by Cincinnati Children’s Hospital Medical Center, with pharmaceutical collaborators, combined liver organoids and donor-matched CD8 T cells to investigate flucloxacillin-associated immune liver injury. The study asked whether an organoid assay that remained uninjured by the drug alone could produce an injury response when the relevant adaptive immune component was added.

The liver organoids were derived from induced pluripotent stem cells generated from healthy donors’ blood cells. Micropatterned hydrogel arrays formed aggregates of approximately 250 cells in 500-μm cavities. Across three iPSC lines, the arrays produced more uniform organoid numbers and diameters than conventional Matrigel domes while retaining comparable albumin secretion and hepatic markers. The initial enrichment medium still contained 2% Matrigel; the array format avoided bulk matrix embedding rather than eliminating every animal-derived matrix component.

Seven-day drug tests without T cells distinguished intrinsic toxicity from the later immune experiment. Chlorpromazine reduced ATP viability and albumin secretion and increased dead-cell staining; one reported IC50 was 14.4 μM, with a three-donor average of 11.12 μM. Flucloxacillin produced no comparable direct toxicity up to 1 mM. Streptomycin also lacked a toxic response at the reference clinical exposure range, although much higher concentrations reduced viability.

Donor Matching Reduced Nonspecific Immune Injury

The investigators compared autologous CD8 T cells with allogeneic cells from a different donor. Allogeneic cultures showed cell death and impaired albumin secretion, especially with anti-CD3/CD28 stimulation and a higher T-cell proportion. This supplied a reason to use donor-matched cells when the aim was to study a drug-associated immune response rather than HLA mismatch.

Autologous cocultures did not show significant overt cell death in this optimization experiment. However, strong nonspecific stimulation at the higher T-cell proportion reduced albumin secretion and increased granzyme B release. The lack of a dead-cell signal therefore did not imply that liver function was unchanged under every immune condition.

Flucloxacillin Priming Produced Strong and Weak Carrier Responses

The T-cell priming cohort included four healthy HLA-B*57:01 carriers and four non-carriers. Dendritic cells exposed to flucloxacillin were used to prime naïve CD8 T cells, followed by expansion and functional characterization. All four carriers showed some response, but only two had a robust increase of more than twofold in proliferation and activation-related measurements. Non-carrier cells did not show a comparable response.

The activation panel included memory and activation markers as well as CD107a, which reflects cytotoxic degranulation. Measuring several features helped distinguish a broadly positive allele association from the stronger effector response seen in selected donors. The donors were not a cohort of patients with documented flucloxacillin-induced liver injury, so these findings describe an experimentally elicited response in healthy-donor cells.

Strong Responders Damaged Their Matched Liver Organoids

For the injury experiment, liver organoids were exposed to 100 μM flucloxacillin for 72 hours before a further three-day coculture with unprimed or drug-primed autologous CD8 T cells. The reported Figure 5 comparison used a 5:1 effector-to-target ratio. Liver-cell death was distinguished from T-cell death by cell labeling and DRAQ7 staining, and cytokeratin 18 release supplied an additional liver-cell-associated injury measurement.

Primed T cells from strong responders 534 and 622 caused approximately fourfold more organoid dead-cell staining than T-cell-free controls, with increased cytokeratin 18, TNFα and granzyme B. Weak carrier responders and non-carrier cells did not produce comparable liver-cell death or cytokeratin 18 release. The five measurements shown in the injury figure were assay repetitions, rather than five independent donors.

In this small donor series, the injury response corresponded to the strength of functional immune priming rather than allele status alone. Connecting the primed T-cell phenotype to matched liver-cell injury offers a way to investigate that heterogeneity. The present experiments establish donor-specific responses to one benchmark drug; their relationship to individual clinical liver-injury outcomes has yet to be measured.