Full-Spectrum Flow Cytometry Analysis Reveals Dynamics of T Cell Function of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia during Continuous Infusion of Blinatumomab
Authors: Zhang Mingying , Zhang Yuanyuan , Qi Peijing , Lin Wei , Wu Ying , Yu Jiaole , Zheng Huyong , Zhang Ruidong 
Published: December 2024

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most prevalent childhood malignancy. T cell-based bispecific monoclonal antibody blinatumomab shows substantial clinical efficacy; however, some children exhibit suboptimal immune responses, leading to reduced effectiveness. T lymphocyte function is considered as one of the critical determinants of blinatumomab's efficacy. This study investigated the dynamics of T cell function during blinatumomab treatment, providing significant theoretical and practical insights for the clinical treatment of pediatric BCP-ALL.

Methods: We will recruit 40 children with BCP-ALL who are identified as high-risk for relapse, either due to minimal residual disease (MRD) ≥1% or high-risk genetic adverse characteristics, following 15 days of routine induction chemotherapy. Subsequently, these children received a continuous infusion of blinatumomab, based on clinical experience with induction chemotherapy. Peripheral blood mononuclear cells (PBMNCs) were collected before the blinatumomab treatment and on days 2, 7, and 14 for patients classified as medium risk, or on days 2, 7, 14, 21, and 28 of high risk. Both groups included children with good and poor responses to blinatumomab. The proportion of CD3+ (total T lymphocytes), CD4+ (T helper lymphocytes), CD8+ (T cytotoxic lymphocytes) and its subsets(naïve T, central memory [TCM], effector memory [TEM], effector memory cells re-expressing CD45RA [TEMRA]), γδT cells, CD4-CD8- T, CD4+CD8+ T, Treg cells, NK cells, NKT cells, and every subsets'expression of the exhaustion molecules (PD-1, TIM-3, LAG-3, TIGIT, CTLA-4, BTLA and CD160) and activation molecules (CD69, CD80, CD86, and Granzyme B) were determined by the full-spectrum flow cytometry with total 23 cell markers in one tube.

Results: Until now, 4 patients (3 with medium risk and 1 with high risk) have been enrolled; they have responded well to blinatumomab treatment, achieving MRD negativity. Patients with medium-risk received a 14-day blinatumomab treatment, while the high-risk underwent a 28-day blinatumomab, following 15 days of routine induction chemotherapy. The percentage of CD3+ T cells declined on day 2 (medium-risk: from 54.92% to 46.29%; high-risk: from 73.94% to 32.51%). Subsequently, the percentage rose above the baseline in both groups (medium-risk: from 46.29% to 90.2% to 86.94%; high-risk: from 32.51% to 89.88% to 94.65% and maintaining this level). The percentage of CD8+ T cells declined on day 2 (medium-risk: from 52.57% to 36.98%; high-risk: from 66.33% to 51.21%). In the medium-risk group, the percentage returned to baseline (from 36.98% to 54.89% to 45.33%), whereas in the high-risk group, it further reduced over subsequent time points. The proportion of CD4+ T cells increased on day 2 in both groups (medium-risk: from 38.56% to 43.45%; high-risk: from 16.75% to 31.03%). It then decreased to baseline on day 7 in the medium-risk group, before increasing again on day 14. In contrast, the percentage continued to ascend at all surveillance time points in the high-risk group, which was different from the trend observed for CD3+ and CD8+ T cells. The percentage of exhausted molecules (Tim3 and PD-1) of CD3+ T cells decreased on day 2 and then arose. The percentage of activated molecules (CD69, CD80, CD86, and Granzyme B) of CD3+ T cells increased on day 2 and then decreased. A similar phenomenon was observed in CD8+ T cells in the medium-risk group. We also noted that Tcm started to decrease on day 7, while Tem and Tex rose instead. The expression of CD69, CD80, and CD86 of Tex arrived at its highest level on day 2, then fell below baseline. Treg cells and monocytes were always above the baseline levels from day 2 to the end of the treatment. No obvious changes were observed in γδT, CD4-CD8- T, CD4+CD8+ T, NK, and NKT cells in the current data.

Conclusion: Our data demonstrated the dynamics of T cell subsets in BCP-ALL during continuous infusion of blinatumomab. The trends of CD3+ T cells and their expression of exhaustion and activation molecules were coincident with CD8+ T cells throughout blinatumomab treatment. The relationship between the dynamics of T cell function and clinical efficacy (complete remission, MRD negative rate) warrants further exploration, which contributes to identifying the predictive biomarker and guiding the precision treatment of clinical immunotherapy. This study is ongoing with a larger sample size.