Scalable generation of polyfunctional therapeutic virus-specific T cells with tissue-homing features and resident-memory-like plasticity across viral targets

Technical Summary: Scalable Generation of Polyfunctional Therapeutic Virus-Specific T Cells

Problem Statement
Adoptive transfer of virus-specific T cells (VSTs) is a proven strategy for controlling opportunistic viral infections in immunocompromised patients. However, broad clinical implementation is hindered by low precursor frequencies of virus-specific T cells (particularly for adenovirus and BK virus), manufacturing constraints, and the need for rapid, scalable production. Furthermore, while the migratory profile of therapeutic VSTs is critical for controlling tissue-resident viral replication, their capacity to acquire tissue-resident memory (TRM)-like features and their specific chemokine receptor profiles remain poorly characterized. Existing protocols often rely on whole leukapheresis, which can lead to the outgrowth of non-specific T cells and natural killer (NK) cells, or require prolonged manufacturing timelines that delay clinical intervention.

Methodology
The authors developed a 10-day, Good Manufacturing Practice (GMP)-compatible expansion strategy utilizing Interferon-γ (IFN-γ)-enriched cells derived from HLA-matched healthy donors. The study compared two starting materials: total peripheral blood mononuclear cells (PBMCs) from leukapheresis and IFN-γ-enriched fractions obtained via magnetic selection (CliniMACS Prodigy).

The expansion protocol involved two distinct conditions to determine the optimal stimulation strategy:

1. Unpulsed (UP) Feeders: Cells were expanded with irradiated autologous feeder cells that were not pulsed with viral peptides.
2. Pulsed (P) Feeders: Cells received a second peptide stimulation during expansion using peptide-pulsed feeders.

The study utilized a diverse panel of viral targets, including Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), Adenovirus (ADV), and BK Virus (BKV). The researchers employed a multi-dimensional characterization approach, including:

• Flow Cytometry: To assess T-cell purity, CD4/CD8 ratios, cytokine production (IFN-γ, TNF, IL-2), cytotoxicity (CD107a), and chemokine receptor expression (CCR1, CCR5, CXCR3, etc.).
• TCRβ Sequencing: To analyze clonality and repertoire diversity.
• RNA Sequencing: To evaluate global transcriptomic profiles and gene set enrichment.
• Functional Assays: Including alloreactivity testing against allogeneic PHA blasts and cryopreservation stability studies.
• Plasticity Induction: A specific subset of experiments involved restimulating thawed VSTs with cognate antigen and TGF-β1 for 3 days to assess the acquisition of TRM markers (CD69 and CD103).
• Scale-Up: A proof-of-concept large-scale manufacturing run (G-REX vessels) was performed for BKV-specific T cells to demonstrate feasibility for multi-dose banking.

Key Results

• Enrichment and Purity: Starting with IFN-γ-enriched fractions significantly improved product purity compared to whole leukapheresis. The UP condition maintained >90% CD3+ T cells and prevented the NK cell outgrowth observed in some leukapheresis-derived expansions.
• Impact of Re-stimulation: Contrary to the hypothesis that a second peptide stimulation would enhance yield, the UP condition (no additional peptide restimulation) proved superior. The P condition skewed the CD4/CD8 ratio toward the dominant subset, reduced the frequency of antigen-responsive cells, and significantly downregulated chemokine receptors (CCR1, CCR5, CXCR3) associated with tissue homing.
• Specificity and Polyfunctionality: Expanded VSTs achieved high antigen specificity (>80% IFN-γ production upon restimulation) even from low initial precursor frequencies. The products displayed virus-directed polyfunctionality (co-production of IFN-γ, TNF, and IL-2) and antigen-specific cytotoxicity without detectable alloreactivity against allogeneic cells.
• Repertoire and Transcriptomics: TCRβ sequencing confirmed clonal focusing during expansion. RNA-seq analysis revealed that the UP condition better preserved activation- and cytokine-related transcriptional programs compared to the P condition, which showed increased adhesion-associated programs but reduced activation markers.
• Tissue-Homing Potential: The expanded VSTs exhibited an effector-memory phenotype (CCR7-/CD45RA-) with broad expression of chemokine receptors (CXCR3, CCR5, CCR2) compatible with trafficking to inflamed peripheral tissues.
• Resident-Memory-like Plasticity: A novel finding was that manufactured VSTs retained the plasticity to acquire TRM features. Upon 3 days of exposure to cognate antigen and TGF-β1, VSTs upregulated CD69 and CD103. Crucially, this phenotypic shift occurred without a loss of antiviral cytokine production or a shift toward an IL-10-dominated regulatory profile.
• Scalability: The protocol successfully scaled to generate >5.5 × 10⁸ BKV-specific T cells with high specificity and polyfunctionality. The product remained stable after cryopreservation for one year and retained the ability to acquire TRM markers post-thaw.

Significance and Claims
The paper claims to establish a rapid, scalable, and GMP-compatible platform for generating allogeneic VST products that are highly specific, polyfunctional, and suitable for cryobanking. Key contributions include:

1. Optimized Manufacturing: Demonstrating that omitting additional peptide restimulation during expansion preserves a balanced CD4/CD8 ratio, maintains antigen responsiveness, and retains chemokine receptor profiles essential for tissue homing.
2. Addressing Low Precursor Frequencies: Validating the strategy for viruses with low memory T-cell frequencies (ADV and BKV), enabling the generation of therapeutic doses from limited starting material.
3. Phenotypic Plasticity: Providing the first evidence that manufactured antiviral VSTs can acquire resident-memory-like features (CD69+/CD103+) in response to TGF-β1 and antigen while maintaining antiviral effector functions. This suggests a potential for enhanced persistence in infected tissues.
4. Clinical Relevance: The use of spare material from compassionate-use manufacturing ensures the findings are grounded in realistic clinical constraints and donor variability.

The authors conclude that this platform supports the development of VST products with tissue-adaptive properties for treating refractory viral complications and serves as a potential starting population for future engineered-cell applications. They acknowledge limitations regarding the lack of in vivo validation and the variability inherent in using clinical donor samples, noting that further studies are required to correlate these in vitro features with in vivo persistence and efficacy.