In recent years, relapsed or refractory hematological malignancies have been treated with chimeric antigen receptor (CAR-)T cells with unprecedented success. There are currently six US FDA-approved CAR-T cell therapies (Table 1) [1], of which four target CD19. Anti-CD19 CAR-T cells are used in B cell malignancies, such as relapsed or refractory follicular lymphoma, large B cell lymphoma (LBCL), mantle cell lymphoma (MCL), and precursor B cell acute lymphoblastic leukemia (ALL). The other two CAR-T therapies target B cell maturation antigen (BCMA) and are approved for the treatment of relapsed or refractory multiple myeloma.

Chimeric antigen receptors (CARs) are synthetic immunoreceptors that combine an antibody-derived antigen-binding extracellular domain with activatory intracellular signaling domains of the CD3/T cell receptor (TCR) complex and T cell co-stimulatory receptors (Fig. 1) [2,3,4]. Thus, T cells can be engineered with CARs to recognize virtually any cell surface antigen. The first generation of CARs possessed the CD3ζ intracellular domain as their sole signaling domain, and this design was able to induce tumor cell killing in vitro but performed poorly in vivo. The addition of a signaling domain from a T cell co-stimulatory receptor (such as CD28 or 4-1BB) marked the second generation of CARs, providing CAR-T cells with enhanced activation, expansion, and persistence in vivo [2, 5,6,7,8]. All currently approved CAR-T cell therapies utilize second-generation CARs, and they target the antigens CD19 and BCMA due to specificity and expression restricted to the B cell lineage and plasma cells, respectively [2, 9]. Further iterations are being studied, such as third-generation CARs containing two co-stimulatory domains, e.g. a combination of CD28 and 4-1BB [6,7,8]. Furthermore, additional tumor-associated target antigens are being evaluated, including CD22, CD33, CD70, CD123, CD138, CD171, HER2, EGFR, B7-H3, claudin 6, gp120, GPRC5D, PSMA, and mesothelin [6, 10,11,12,13,14]. In attempts to reduce tumor escape through antigen loss, simultaneous targeting of multiple antigens, such as a combination of CD19 and CD22, has also been considered [15, 16].

Structure of CAR-T cells. Created with BioRender.com

Despite favorable clinical response rates, the manufacturing of CAR-T cells is as complex as for any other adoptive cell therapy, resulting in logistical challenges and high cost of these treatments [2]. Leukapheresis is performed on the patients to isolate autologous leukocytes, from which T cells are enriched in the manufacturing facility. In a process spanning two to four weeks, these T cells are activated, retrovirally or lentivirally transduced with the CAR gene, and expanded to yield sufficient doses of CAR-T cells to re-infuse into the patient after conditioning chemotherapy. In addition to logistics, costs and turnaround time, the multifaceted impacts of CAR-T cells on the human body have to be taken into consideration [19, 20]. In this review, we will discuss our current understanding of pathophysiology and management strategies for the major and most frequent CAR-T cell-related adverse events: Cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and hemophagocytic lymphohistiocytosis or macrophage activation syndrome (HLH/MAS).

Since many mechanistic aspects of their pathophysiology are still poorly understood, the diagnosis and treatment of CAR-T cell-related adverse events pose unique challenges. These adverse effects span a broad range of severities and manifestations and may involve multiple organ systems (Fig. 2), similar to immune-related adverse events (irAEs) that are known to occur upon use of immune checkpoint inhibitors [21, 22]. On-target off-tumor effects, whereby healthy cells expressing the target antigens are attacked by the CAR-T cells, are common, but in the cases of therapies targeting CD19 and BCMA they are manageable and well-tolerated [20]. There have been reports of allergic reactions and metabolic abnormalities such as tumor lysis syndrome after CAR-T cell infusion [20, 23,24,25]. However, CRS, ICANS, and HLH/MAS are regarded as the most dominant CAR-T cell-related toxicities.

Simplified CAR-T therapy’s adverse effects. Created with BioRender.com

CRS and ICANS are the most frequent adverse events of CAR-T cell therapies [26,27,28,29,30,31]. In clinical trials of the six currently US FDA-approved CAR-T products, CRS had an incidence of between 49 and 95%, with 1–24% for grade ≥ 3 CRS. ICANS occurred in 12–60% of the patients, with grade ≥ 3 ICANS in 3–50% (Table 2) [26,27,28,29,30,31]. Differences in grading systems likely contributed to the large variability in recorded frequencies of adverse events between clinical trials, making it difficult to draw direct comparisons. Nevertheless, a common pattern is the earlier median onset of CRS within the first week after CAR-T cell infusion, compared to ICANS which tends to also have a longer average duration (Fig. 3) [26,27,28,29,30,31]. Despite the high incidence of CAR-T cell-related adverse events, a meta-analysis estimated treatment-related death at only 1% [36].

Median onset and duration of CRS and ICANS after six FDA-approved CAR-T cell therapies

Similarly, an initial survey found an incidence of only 3.48% for HLH/MAS after CAR-T cell therapy between 2016 and 2018 [21]. However, more recent phase I clinical trials of anti-CD22 CAR-T cells reported 32.7% and 35.6% of patients developing HLH, respectively [17, 18]. These findings, coupled with the high morbidity and mortality associated with this syndrome, are drawing increasing attention towards CAR-T cell-related HLH/MAS [21].

CRS, ICANS, and HLH/MAS are understood to be the consequences of CAR-T cell activation in response to tumor recognition, leading to the excessive release of cytokines and danger signals, though this phenomenon is not unique to CAR-T cell therapies and also occurs in other immunotherapies [32,33,34,35].

CRS is a clinical syndrome that affects multiple organ systems and usually starts from generalized symptoms or signs, such as fever, fatigue, tachycardia, and myalgias. The fever can exceed 105°F/40.5 °C [37]. More severe CRS can present as hypotension, hypoxia, capillary leak syndrome, multiple organ failures, disseminated intravascular coagulation (DIC), and even HLS/MAS [33]. Circulating inflammatory cytokines increase the vascular permeability and third-spacing of fluid, which mimics sepsis but usually with neutropenia [33, 34, 38]. According to the severity of the clinical presentation, CRS can be separated into mild CRS and severe CRS. Constitutional symptoms and/or grade ≤ 2 organ toxicity indicate mild CRS, and severe CRS is characterized by grade ≥ 3 organ toxicity with potentially life-threatening consequences [39,40,41].

Multiple cytokines are elevated after CAR-T cell infusion, such as interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), granulocyte–macrophage colony-stimulating factor (GM-CSF), and interleukin 6 (IL-6), but cytokine levels are not always correlated with CRS severity, and their timely monitoring is challenging [5, 39, 41,42,43,44,45]. After antigen binding, CAR-T cells release large amounts of cytokines and perforin/granzymes, which are essential for anti-tumor activity. In addition to caspase 3 activation in the target cells, granzyme A and granzyme B were found to cleave gasdermin D (GSDMD) and E (GSDME), respectively, which are hallmarks of pyroptosis [47,48,49,50]. In contrast to apoptosis, which is a non-inflammatory programmed cell death pathway, pyroptosis is a highly inflammatory form of cell death. Cleaved gasdermins release their N-terminal domains, which can insert into the cell membrane and form pores, resulting in the release of pro-inflammatory factors from the dying cells. Thus, high expression of GSDME will lead to preferential pyroptosis, despite both apoptosis and pyroptosis being caspase-mediated [51]. This is consistent with the finding that high GSDME expression is associated with severe CRS [47].

Pyroptotic cells release large amounts of damage-associated molecular patterns (DAMPs), which include heat shock proteins (HSPs) and high­mobility group box 1 (HMGB1) and activate the innate immune system. Here, macrophages and other myeloid cells play a critical role in the pathogenesis of CRS (Fig. 4) [52,53,54]. HMGB1 binds Toll-like receptor 2 (TLR2) and TLR4 on these cells, activating the interferon regulatory factor (IRF), mitogen-activated protein kinase (MAPK), and NFκB pathways [46]. These pathways trigger the release of systemic cytokines, including interferons, IL-1β, and IL-6 [46, 47, 52, 53]. Activation of TLR2 induces not only the expression and secretion of IL-6 but also the generation of soluble IL-6 receptor (sIL-6R), which enhances the pro-inflammatory properties of IL-6 [55].

Basic pathophysiology of CRS. Created with BioRender.com

In summary, CAR-T cells, which are designed to achieve high anti-tumor efficacy through combinations of T cell-activating signaling domains and high-affinity antigen recognition, inevitably cause the secretion of a large amount of perforin and granzymes [46, 56, 57]. This supraphysiological response may cause excessive pyroptosis, initiating a cascade that leads towards CRS.

IL-6 is well known for its pleiotropic function, including the involvement in B cell and T cell differentiation, bone homeostasis, production of acute-phase proteins, and chronic inflammatory processes in vascular endothelial cells [62, 63]. Early studies in glucocorticoid-resist