Background and Aims This study developed and validated a novel immune status evaluation system utilizing multiplex cytokine profiling to objectively assess post-CD19 CAR-T therapy immune responses and facilitate early identification of cytokine release syndrome (CRS). Particularly by quantifying the effects of various cytokines and proposing a formulaic algorithm, it provides an effective tool for the simplified, scientifically rigorous, and rapid assessment of high-volume data.

Methods The specimens were from 111 patients with refractory/relapsed B-cell acute lymphoblastic leukemia (B-ALL) treated with CD19 CAR-T therapy at Lu Daopei Hospital since March 2020. The cohort comprised a training set (n=34) for developing a cytokine-based scoring system and a validation set (n=77) to evaluate clinical concordance. Serial measurements of 24 cytokines (IFN-γ, IL-1β, IL-2, IL-6, IL-10, IL-12p70, TNFα, TNFβ, IL-4, IL-5, IL-8, IL-17A, IL-17F, IL-22, IL-2RA, MCP-1, GM-CSF, IL-15, Granzyme B, REG3a, ST-2, TNFRI, Elafin and MIP-1α) were performed in the training set at six timepoints: pre-CAR-T treatment (day 0), and days 4, 7, 14, 21, and 28 post-treatment. The validation set underwent cytokine profiling at days 0 and 7.

Results Our study showed that peak cytokine levels occurring at 7-10 days post-treatment, followed by progressive decline as CAR-T/tumor cell interactions diminished and tumor clearance advanced. This reduction coincided with decelerated expansion of CAR-T, CD3+, and CD8+ T-cell populations, with cytokine concentrations returning to near-baseline by approximately day 30.

For the convenience of clinical application, we developed a cytokine scoring system for immune status assessment. The 24 factors were divided into four weight levels based on their contribution to predicting CRS: Class I cytokines (IFN-γ, IL-2, IL-6, IL-10, ST-2, IL-8, GM-CSF) weighted at 2.0; Class II (IL-2RA, IL-17F, REG3a, IL-1β, MCP-1, TNFRI) at 1.0; Class III (IL-4, IL-5, IL-22, IL-15, IL-12p70) at 0.5; and Class IV (Elafin, TNFα, Granzyme B) at -1.0. A cytokine is assigned a weight point if its peak value exceeds three times the threshold. The results for each cytokine are summed to obtain a total score. Following targeted therapy, an immune status score ≤ 8 points is classified as CRS Grade 0-1; a score > 8 points and < 18 points is classified as CRS Grade 2; and a score ≥ 18 points is classified as CRS Grade 3-4. Clinical validation demonstrated overall sensitivity of 89.61%, specificity of 96%, positive predictive value (PPV) of 95.83%, and negative predictive value (NPV) of 90%. Crucially, for distinguishing Grade 0-1 CRS (outpatient-manageable) from ≥Grade 2 CRS (requiring hospitalization per ASTCT criteria), the system achieved enhanced performance: 91.55% sensitivity, 99% specificity, 98.48% PPV, and 92% NPV, confirming its utility in guiding critical clinical decisions.

Conclusions This standardized system provides an objective, high-throughput tool for immune monitoring and early intervention, with significant implications for clinical decision support in cellular immunotherapy.

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2025
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