Background B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common malignancy in children. Blinatumomab is a bispecific T-cell engager (BiTE) antibody targeting CD3 and CD19. T-cell function is considered a critical factor influencing the efficacy of Blinatumomab. This study analyzed functional dynamics in peripheral T cells of newly diagnosed BCP-ALL patients during Blinatumomab treatment using full-spectrum flow cytometry, aiming to identify immune patterns linked to treatment outcomes and support personalized immunotherapy optimization.

Methods The study enrolled pediatric BCP-ALL patients from the CCLG-ALL 2018 protocol, including intermediate-risk (MR, day 15 MRD 1-10%) and high-risk (HR, MRD ≥10% or high-risk genetics) groups. PBMNCs were collected at baseline and multiple time points (days 2, 7, 14 for MR; plus days 21, 28 for HR). A 23-color full-spectrum flow cytometry panel analyzed 23 T-cell subsets and expression of 4 activation (CD69, CD80, CD86, Granzyme B) and 7 exhaustion markers (PD-1, TIM-3, LAG-3, TIGIT, CTLA-4, BTLA, CD160).

Results A total of 42 pediatric BCP-ALL patients treated with blinatumomab were included in the analysis (28 MR and 14 HR). Full-spectrum flow cytometry was performed at 185 time points. The median age was 6 years (range: 1–15), and 59.5% (25/42) were male. High-risk genetic alterations were present in 64.3% (27/42) of patients. The median bone marrow (BM) blast burden at enrollment was 5.51% (range: 0.02%–50.38%).

Following treatment, all 14 HR patients and 21 MR patients achieved deep MRD negativity, while 7 MR patients failed to reach deep molecular remission (NGS MRD ≥10⁻⁶). On day 2, CD3⁺ T-cell proportions declined significantly in both risk groups before gradually increasing and eventually surpassing baseline levels. Notably, MR patients who failed to achieve deep remission showed a more pronounced drop in CD3⁺ T cells on day 2, suggesting lower baseline T-cell counts or impaired functionality, potentially leading to early exhaustion or rapid recruitment to leukemia sites. Importantly, the dynamic trend of CD3⁺ T cells closely mirrored that of CD8⁺ T cells, both showing early depletion followed by gradual recovery. This similarity likely reflects the dominant contribution of CD8⁺ cytotoxic T cells within the total CD3⁺ T-cell population during the early phase of blinatumomab-induced immune activation, especially given that CD8⁺ cells are primary mediators of cytotoxicity.

Treatment-responsive patients exhibited higher baseline levels of central memory (Tcm) and effector memory (Tem) T cells, while non-responders had significantly lower levels, suggesting that pre-existing memory T-cell pools may facilitate more rapid and effective immune engagement. Effector T cells (Teff) increased sharply on day 2 in responders, then declined toward baseline. In contrast, non-responders showed delayed Teff activation (day 7), indicating suboptimal immune priming. CD4⁺ T cells and NK cells increased slightly on day 2 and then stabilized. In MR patients, Treg levels rose progressively, but with smaller increases in those achieving deep remission, suggesting that excessive immunosuppression may impede response. In HR patients, Tregs peaked on day 14 and subsequently declined, indicating transient immunosuppression followed by immune rebalancing.

Notably, expression of activation markers (CD69, CD80, CD86) and Granzyme B on CD3⁺ and CD8⁺ T cells peaked on day 2 and then declined to near-baseline levels, further confirming that T cells were rapidly activated to exert anti-tumor effects, followed by a regulatory phase to maintain immune homeostasis. NK and NKT cells maintained high Granzyme B expression throughout treatment, suggesting sustained cytotoxic potential and indirect activation. Meanwhile, MDSCs and monocytes remained elevated, indicating a persistently immunosuppressive microenvironment.

ConclusionsThis study reveals the complex dynamic changes of T-cell subsets during Blinatumomab treatment and identifies multiple immune markers closely associated with therapeutic efficacy. Early alterations in T-cell subsets, including activation and exhaustion markers, can serve as potential indicators for predicting treatment response and assessing immune status. These findings provide important references for efficacy monitoring and optimization of personalized immunotherapy strategies in pediatric BCP-ALL.

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2025
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