Efficient generation of chimeric antigen receptor (CAR) T cells is highly influenced by the quality of apheresed T cells. Healthy donor-derived T cells usually proliferate better than patients-derived T cells and are precious resources to generate off-the-shelf CAR-T cells. However, relatively little is known about the determinants that affect the efficient generation of CAR-T cells from healthy donor-derived peripheral blood mononuclear cells (PBMC) compared with those from the patients’ own PBMC. We here examined the efficiency of CAR-T cell generation from multiple healthy donor samples and analyzed its association with the phenotypic features of the starting peripheral blood T cells. We found that CD62L expression levels within CD8⁺ T cells were significantly correlated with CAR-T cell expansion. Moreover, high CD62L expression within naïve T cells was associated with the efficient expansion of T cells with a stem cell-like memory phenotype, an indicator of high-quality infusion products. Intriguingly, genetic disruption of CD62L significantly impaired CAR-T cell proliferation and cytokine production upon antigen stimulation. Conversely, ectopic expression of a shedding-resistant CD62L mutant augmented CAR-T cell effector functions compared to unmodified CAR-T cells, resulting in improved antitumor activity in vivo. Collectively, we identified surface expression of CD62L as a concise indicator of potent T cell proliferation. CD62L expression is also associated with functional properties of CAR-T cells. These findings are potentially applicable to selecting optimal donors to massively generate CAR-T cell products.

Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.

Background: This study was conducted to investigate the short-term efficacy and safety of rhTPO for the management of severe ITP in the elderly as first-line treatment. Methods: A total of 54 elderly patients with severe ITP were studied, including 39 patients treated with a combination regimen of rhTPO plus standard treatment (glucocorticoid; rhTPO group) and 15 patients treated with glucocorticoid treatment alone (control group). The response rate, time to initial response, peak platelet counts, and time to peak platelet counts were compared, and clinical characteristics correlated with the efficacy of rhTPO were analyzed. The efficacy of rhTPO in the elderly is comparable to the non-elderly in terms of the OR, CR, time to initial response, and peak platelet counts. Results: There were no differences in the overall response (OR) and the complete response (CR) in the rhTPO group compared to the control group. The time to initial response in the rhTPO group was shorter than that in the control group (p = 0.032). In patients without intravenous immunoglobulin (IVIg) and platelet transfusion, the peak platelet counts in the rhTPO group were higher than those in the control group (p = 0.003). Conclusions: Standard glucocorticoid treatment plus rhTPO effectively shortens the time to response and increases platelet counts in the elderly with severe ITP.

Providing cytokine signaling is a key strategy to boost the efficacy of chimeric antigen receptor (CAR) T-cell therapy. However, the individual roles of key downstream mediators, STAT3 and STAT5, remain incompletely understood. In this study, we engineered CAR T cells to express constitutively active mutants of STAT3 (Y640F; caSTAT3) and STAT5 (N642H; caSTAT5) to investigate their individual functions. In vitro, caSTAT3 CAR T cells exhibited enhanced effector function and a robust memory phenotype, with broader transcriptional changes involving both effector- and memory-associated genes compared with caSTAT5 CAR T cells. However, caSTAT3 CAR T cells failed to expand because of activation of apoptosis-related gene programs. In contrast, caSTAT5 CAR T cells demonstrated sustained proliferation over time. Despite the limited in vitro expansion, caSTAT3 CAR T cells exhibited reduced transgene toxicity in vivo and exerted durable antitumor activity in both leukemia and solid tumor models without significant off-tumor toxicity. Titrated expression of caSTAT3 maintained enhanced effector function without inducing apoptosis. On the other hand, caSTAT5 CAR T cells efficiently accumulated in tumors but also infiltrated nontumor tissues, causing lethal systemic toxicity. Co-expression of caSTAT3 and caSTAT5 markedly enhanced the long-term proliferative capacity of CAR T cells, even in the absence of antigen stimulation or cytokine supplementation. These findings elucidate the distinct impacts of STAT3 and STAT5 activation on CAR T-cell behavior and suggest that selective activation of STAT3 at optimal levels may improve CAR T-cell efficacy while minimizing off-tumor toxicities.

Supplementary Table S2. Guide RNA sequences used in this study.

Differential pathway activity between caSTAT3+5 and caSTAT3 CAR-T cells.