Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy

Success of adoptive T cell therapy (ATT) is dependent on sufficient numbers of T cells and the characteristics of the final T cell product. In several studies, clinical grade CD19 CAR T cell products could not be generated from about 6-30% patients, particularly if they were isolated from older or heavily pretreated diffuse large B cell lymphoma (DLBCL) patients. In cyclophosphamide/fludarabine-lymphodepleted patients with persistent or progressive disease a sequential second dose of T cells has been shown to be effective resulting in tumor regression. Here we investigated to what extend T cell numbers could be increased via prolonged expansion with standard cytokines IL-7/IL-15 and how transcriptome and function of central memory T cells (Tcm) longitudinally change during culture.

Method:

Murine and human T cells were cultured with the cytokine combination IL-7/IL-15. Short-term expanded (ST, one week) and long-term expanded (LT) CD8⁺ (4 weeks) and CD4⁺ (3 weeks) T cells were compared for proliferation capacity (CFSE), extent of apoptosis (AnnexinV), up-regulation of T cell inhibitory receptors (TIRs) and cytokine expression pattern after in vitro re-stimulation upon anti-CD3/CD28 stimulation. Further, RNA sequencing of ST and LT expanded murine CD8⁺ and CD4⁺ Tcm followed by unsupervised hierarchical clustering, principal component analysis (PCA) and differential expression analysis was performed. In vivo mouse models were used to analyze engraftment, persistence and anti-tumor capacity applying our bioluminescent dual-luciferase reporter mouse (BLITC - bioluminescent imaging of T cells) allowing us to monitor migration, expansion (RLuc luciferase) and activation (NFAT-driven Click-beetle luciferase) of adoptively transferred T cells in vivo. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors and DLBCL patients.

Results:

There was a 50-fold increase of T cells in LT vs. ST culture, the Tcmproportion was extended and stem cell markers were comparable or even higher expressed in LT expanded T cells. Differential analysis revealed 2786 (CD8) and 912 (CD4) with statistically significant expression alterations with generally only moderate effect size when comparing LT and ST expanded T cells. Interestingly, the dynamically modified genes largely overlapped for CD8 and CD4 T cells suggesting culture-associated changes. Comparable RLuc signals and T cells counts in peripheral lymph nodes (LN) and spleen indicate similar engraftment (4 weeks post ATT) and persistence capacities (up to 6 months post ATT) of transferred ST and LT T cells. SV40-TAg⁺ tumor bearing mice were treated with TCR-I retrovirally transduced CD8⁺ BLITC T cells, which were ST or LT expanded. The T cells infiltrated rapidly in the tumor where they got similarly activated resulting in a complete tumor rejection in all recipient mice.

Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors (n=3-5) and DLBCL patients (n=3) who were eligible for CAR T cell therapy. LT T cell expansion from healthy donors resulted in a 10.000-fold increase of CD8⁺CD45RO⁺CCR7⁺ T cells. In vitro assays showed comparable apoptosis and expression of TIRs between ST and LT CD8 T cells and stable expression of IFN-g and TNF-a within the first 3 weeks. The CD8⁺CD45RO⁺CCR7⁺ T cell expansion from DLBCL patients was weaker in comparison to healthy donors. The extent of cell death and up-regulation of TIRs after re-stimulation was comparable between ST and LT T cells, whereas cytokine expression varied individually.

Conclusion:

Our data suggest that it is feasible to expand CD8⁺ and CD4⁺ murine and human T cells up to a month, thereby increasing numbers of T cells with Tcm/Tscm properties and with sustained function for murine and human T cells from healthy donors, whereas there seems to be a high individual variance for DLBCL patients, which warrants further investigation in larger patient cohorts.