{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T09:42:33Z","timestamp":1775986953163,"version":"3.50.1"},"posted":{"date-parts":[[2025,4,1]]},"group-title":"Cancer Biology","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,4,1]],"date-time":"2025-04-01T00:00:00Z","timestamp":1743465600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,4,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Chimeric Antigen Receptor T-cell (CAR-T) therapy targeting CD19 has transformed the treatment of relapsed\/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the phenotypic heterogeneity of CAR+ T-cells and their interactions with the immune system remain poorly understood. Here, we characterize CAR+ T-cell subsets and persistence in patients receiving standard-of-care tisagenlecleucel in an effort to optimize therapeutic efficacy in pediatric BCP-ALL.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>Nineteen pediatric patients with relapsed\/refractory BCP-ALL treated with tisagenlecleucel in Poland were included in the study. CAR+ T-cell composition and the peripheral blood mononuclear cell (PBMC) immune system were assessed using mass cytometry, with qPCR validation of CAR+ T-cells. Machine learning algorithms in R classified phenotypes. Statistical analyses examined associations between CAR+ T-cell subsets, immune system changes, and CAR-T therapy outcomes, including cytokine release syndrome (CRS).<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>Infusion product analysis showed a predominance of CD4+ Central Memory (16.6%), MAIT\/NKT (7.04%), and Treg memory (63.3%) cells, with high consistency between paired products. Post-infusion, CAR+ Treg memory cells declined to &lt;1%, while CD8+ subsets expanded. PBMC immune system analysis revealed increased monocyte and NK cell counts post-infusion. Patients who experienced CRS had fewer classical monocytes at day 7 and fewer transitional monocytes at day 28. Early and late NK cells increased post-infusion in all patients. However, pre-infusion levels were lower in patients experiencing CRS. CRS correlated with higher CAR+ CD8+ Terminal Effector and MAIT\/NKT frequencies in infusion products. While CAR+ composition was not linked to disease burden, prior treatments, or therapy outcomes, changes in monocyte and NK cell dynamics were associated with disease burden, relapse, and CRS.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>This study provides a detailed characterization of CAR-T CD19 cell composition and post-infusion dynamics in pediatric BCP-ALL patients treated with tisagenlecleucel. Infusion products were predominantly CAR+ CD4+ T cells with a Central Memory phenotype, including a notable Treg memory subset that declined post-infusion. CAR+ CD8+ subsets expanded, coinciding with immune system shifts. These changes correlated with clinical responses, including CRS, highlighting the role of the immune system in CAR-T therapy outcomes and informing strategies to optimize treatment.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Summary box<\/jats:title>\n                  <jats:sec>\n                    <jats:title>WHAT IS ALREADY KNOWN ON THIS TOPIC<\/jats:title>\n                    <jats:p>CD19-targeted CAR-T therapy has revolutionized the treatment of relapsed\/refractory pediatric BCP-ALL. While studies have examined the diverse composition of CAR-T infusion products in adult large B-cell lymphoma (LBCL), there is limited data in the pediatric BCP-ALL setting.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>WHAT THIS STUDY ADDS<\/jats:title>\n                    <jats:p>This study demonstrates that standard-of-care CAR-T infusion products (tisagenlecleucel) are primarily composed of CAR+ CD4+ Central Memory and regulatory T cells, which decline post-infusion as CAR+ CD8+ subsets expand. Additionally, monocyte and NK cell dynamics are closely linked to cytokine release syndrome (CRS) and treatment outcomes, emphasizing the critical role of immune system interactions in CAR-T therapy efficacy.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICY<\/jats:title>\n                    <jats:p>These findings indicate that monitoring immune system shifts could enhance CRS risk assessment and improve patient stratification, leading to more personalized CAR-T therapy. Additionally, insights into CAR+ T-cell composition may guide the development of strategies to optimize CAR-T efficacy, potentially improving long-term treatment outcomes in pediatric BCP-ALL.<\/jats:p>\n                  <\/jats:sec>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.27.645594","type":"posted-content","created":{"date-parts":[[2025,4,2]],"date-time":"2025-04-02T01:24:12Z","timestamp":1743557052000},"source":"Crossref","is-referenced-by-count":1,"title":["Single-cell profiling of CAR-T CD19 cell phenotypes and immune system dynamics in pediatric BCP-ALL"],"prefix":"10.64898","author":[{"given":"A.","family":"Oszer","sequence":"first","affiliation":[{"name":"Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Lodz, Poland"},{"name":"Division of Hematology, Oncology, Stem Cell Transplant, and Regenerative Medicine, Department of Pediatrics, Stanford University, Stanford, CA, 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