Unveiling the “second wave”: Viral-triggered biphasic CAR-T expansion drives late-onset IEC-HS: Characterization and successful management

Abstract Background: While cytokine release syndrome (CRS) and ICANS are well-defined acute toxicities of CAR-T cell therapy, the landscape of late-onset complications is still evolving. A distinct and life-threatening hyper-inflammatory syndrome, now recognized as Immune Effector Cell-associated Hemophagocytic Lymphohistiocytosis-like Syndrome (IEC-HS), can emerge following a period of relative quiescence. The underlying immunological drivers and optimal management of this late toxicity, often triggered by a biphasic CAR-T expansion, remain poorly characterized. Methods: We retrospectively analyzed a cohort of 79 patients receiving CAR-T cell therapy at our institution over the past year. Cases of late-onset IEC-HS were identified based on established HLH diagnostic criteria, coupled with evidence of marked, delayed (secondary) CAR-T cell expansion via flow cytometry. We investigated the incidence, timing, potential triggers, and management outcomes. To dissect the underlying mechanisms, high-dimensional spectral flow cytometry was employed to characterize the immunophenotype of re-expanding CAR-T cells and the broader immune landscape in affected patients. Results: Among 79 patients, 14 (17.7%) developed late-onset IEC-HS, with a median onset of 32 days (range: 15-60) post-infusion. The primary trigger was identified as pathogen-mediated CAR-T cell reactivation, predominantly due to viral infections (e.g., CMV), rather than spontaneous antigen-driven proliferation alone. High-dimensional immune profiling revealed that this secondary expansion was associated with a profound phenotypic shift within the CAR-T cell compartment. Compared to the initial expansion peak dominated by terminally differentiated effectors, the re-expanding CAR-T cells exhibited a less-differentiated, stem-like memory phenotype (e.g., TCF1-positive), suggesting a potent proliferative potential. A risk-stratified approach using low-dose etoposide (50-100 mg per dose), tailored to the patient's proliferation kinetics and clinical severity, effectively controlled the hyper-inflammation in over 90% (13/14) of cases. Notably, patients with baseline CD4+ T-cell dysfunction exhibited recurrent viral infections leading to multiple episodes of CAR-T reactivation, characterized by markedly elevated CXCL10 and IL-18 levels, presenting substantial clinical management challenges. Conclusion: Late-onset, infection-triggered IEC-HS represents a distinct and severe complication of CAR-T therapy, driven by the biphasic expansion of a phenotypically less-differentiated and highly proliferative CAR-T cell population. Alongside aggressive antiviral therapy, controlling the aberrant CAR-T cell proliferation is a critical step in managing IEC-HS. For this, judicious and patient-tailored administration of low-dose etoposide proved to be a highly effective strategy. Impaired CD4+ T-cell immunity is a critical risk factor, creating a permissive environment for the viral triggers of IEC-HS. These findings highlight the need for vigilant infection surveillance and suggest that strategies to restore T-cell function may be crucial for preventing this life-threatening toxicity.