Single-Cell RNA-Sequencing Identifies TCF1 and BCL11B Upregulation As Predictors of Blinatumomab Efficacy in B-Cell Acute Lymphoblastic Leukemia Patients

Background:

Blinatumomab, a CD3/CD19 bispecific monoclonal antibody, has achieved encouraging results in relapsed refractory B-ALL patients. Compared with chemotherapy, it achieves earlier and deeper remission and provides more patients with opportunities for allogeneic hematopoietic stem cell transplantation. However, there are still 30-40% of patients with poor response who cannot benefit from blinatumomab. Studies have shown that blinatumomab’s anti-leukemia effect highly relies on T cell function. However, there are no specific indicators of T cell immunity currently available to predict the efficacy of blinatumomab in clinical.

Aims:

To investigate the mechanism of blinatumomab in treating B-ALL patients using single-cell RNA-sequencing (scRNA-seq), and identify the specific T cell subsets that exhibit anti-leukemia effects.

Methods:

Relapsed refractory B-ALL patients undergoing blinatumomab treatment were included in this study. Peripheral blood samples were collected from 7 B-ALL patients with good response before treatment and 14 days after initiating blinatumomab. CD3+ T cells were isolated by immunomagnetic beads for scRNA-seq. Additionally, the expression of key genes was measured in 35 B-ALL patients (25 cases with favorable response and 10 with poor response) before and on day 3, 7, and 14 of blinatumomab treatment using multi-colored flow cytometry.

Results：

scRNA-seq data revealed significant changes in CD8+ T cell subpopulations following blinatumomab treatment, the proportions of naïve T cells, effector memory T cells (Tem), short-term effector cells (SLEC), and exhausted T cells (Tex) dramatically decreased. On the contrary, the proportions of memory stem cells (T_SCM), central memory T cells (Tcm), effector T cells (Teff), and tissue-resident T cells (Trm) significantly increased.

The enrichment analysis of differentially expressed genes among different cell types showed that TCF7 was highly expressed in naïve T, Tcm and Tscm, while downregulated in SLEC, Tex and Trm. BCL11B expression was low in Tex and Trm, but predominantly found in Tscm, Tcm and Teff. Additionally, functional enrichment in biological processes indicated that TCF7 was mainly involved in differentiation and immune response, while BCL11B was associated with T cell differentiation and apoptosis. A significant positive correlation was found between TCF7 and BCL11B before and after blinatumomab treatment (R = 0.78 P < 0.0001; R = 0.67 P < 0.0001). Following blinatumomab treatment, T cells co-expressing TCF7 and BCC11B transitioned from naïve T cells and Tem to TCM and Teff with high IL7R expression (Teff-IL7R^hi). Notably, SLECs with BCL11B+TCF1- that increased significantly pretreatment gradually decreased after 14 days of blinatumomab, and was replaced by a large amount of Teff with BCL11B+TCF1+, especially Teff-IL7R^hi. The proportion of TCF7+BCL11B+CD8+ T cells slightly increased in Trm after treatment compared with baseline. Furthermore, cell fate trajectory analysis confirmed the differentiation from Trm to Teff and Tex during blinatumomab treatment.

FCM analysis of dynamic changes of TCF1+BCL11B+ T cells in CD8+ T cells showed that their proportion was significantly lower in Tex compared to non-Tex, consistent with the scRNA-seq results. Prior to blinatumomab treatment, the proportion of TCF1+BCL11B+CD8+ T cells in B-ALL patients was significantly lower than in healthy control, especially in poor responders. After 14 days of treatment, the proportion of TCF1+BCL11B+CD8+ T cells in patients with good response was significantly increased (P < 0.0001), approaching normal levels, while it decreased further in poor responders (P = 0.0014), and was significantly lower than that in good responders (P < 0.0001).

Conclusion：

Blinatumomab promotes the differentiation of T cell subsets and exerts anti-leukemia effects in B-ALL patients. The observed upregulation of TCF1 and BCL11B indicates a robust and lasting anti-leukemia effect, which can serve as early indicators of blinatumomab efficacy.

Keywords: B-ALL patient, CD8+T cells, TCF1, BCL11B, Immune response